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Division of Nanomedicine and Nanobiology, National Center for Nanosciences and Technology of China, Beijing, China (F.L., J.M., Y.Z., C.C., X.-J.L.); Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland (J.-J.Y.); Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China (J.M., Y.Z., G.X., X.G., B.S., C.C.); Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas (P.P.F.); and Laboratory of Molecular Imaging, Department of Radiology, Howard University, Washington, DC (P.C.W.)
Received for publication April 28, 2008.
Accepted for publication July 15, 2008.
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Abstract |
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; Chen et al., 2005). Endohedral metallofullerenol nanoparticles ([Gd@C82(OH)22]n) could efficiently inhibit the proliferation of tumors and decrease the activities of enzymes related to reactive oxygen species (ROS) generation in vivo, but the molecular mechanism is still unclear (Wang et al., 2006). ROS such as superoxide radical anion, hydrogen peroxide, singlet oxygen, and hydroxyl radicals have been implicated in the etiology of a wide range of acute and chronic human diseases, including amyotrophic lateral sclerosis, arthritis, cancer, cardiovascular disease, and several neurodegenerative disorders (Valko et al., 2007). Accordingly, species that have a strong capacity for scavenging ROS are of great significance in biomedicine. The ability to functionalize the nanosurfaces of fullerenes and fullerenol nanoparticles presents an opportunity to increase the payload of the ROS scavenger to target cells and tissues.
By using water-soluble derivatives, many investigations have measured the biological significance of fullerenes and their derivatives as prospective nanomedicines. Dugan et al. (1996) demonstrated that carboxylic acid fullerene derivatives had a potent ROS-scavenging activity and that they prevented apoptosis of cultured cortical neurons induced by exposure to N-methyl-D-aspartate agonists. The same derivatives protected the nigrostriatal dopaminergic system from iron-induced oxidative injury and showed effective neuroprotective antioxidant activity in vitro and in vivo. The carboxylic acid fullerene was hundreds of times more protective than vitamin E. A polyethylene glycol-conjugated-fullerene (C60) strongly induced tumor necrosis without any damage to the overlying normal tissue in vivo, making it an excellent candidate for targeted tumor therapy (Tabata et al., 1997). The biological effect of fullerene derivatives has been demonstrated in numerous systems, including reduction in injury after ischemic reperfusion of the intestine (Lai et al., 2000), protection of cells from undergoing apoptosis (Hsu et al., 1998; Bisaglia et al., 2000), reduction in free radical levels in organ perfusate (Chueh et al., 1999), and neuroprotective effects (Dugan et al., 1997; Lin et al., 1999). The potent biological activity of fullerenols has been attributed to a combination of their unique chemical and physical characteristics. Fullerenols may therefore be particularly valuable candidates as respective nanomedicines in biological systems (Chiang et al., 1995; Tang et al., 2007b).
Endohedral metallofullerenes [i.e., compounds in which a fullerene encapsulates a metal atom(s)] have shown great promise for use in biomedical science. Although C60 has been the most commonly studied fullerene in biological systems, few endohedral materials have been synthesized using C60 as a cage molecule because of the limited interior volume of C60. Most endohedral metallofullerenes are synthesized using C82 or higher molecular weight fullerenes, and many derivatives of C82 fullerenes have been synthesized in our laboratory. Gd@C82 is one of the most important molecules in the metallofullerene family (Tang et al., 2007a). Gd@C82(OH)22 is a functionalized fullerene with gadolinium, a transition metal of lanthanide family, trapped inside a fullerene cage, and it was originally designed as a magnetic resonance imaging contrast agent for biomedical imaging (Anderson et al., 2006). We have previously reported that the chemical and physical properties of Gd@C82(OH)x are dependent on the number and position of the hydroxyl groups on the fullerene cage (Tang et al., 2007b).
In this study, electron spin resonance (ESR) spin trap technique is used to provide direct evidence that [Gd@C82(OH)22]n nanoparticles can efficiently scavenge different types of ROS, including superoxide radical anion (
), hydroxyl radical (HO.), and singlet oxygen (1O2), and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH.). In vitro studies using liposomes prepared from bovine liver phosphatidylcholine revealed that [Gd@C82(OH)22]n nanoparticles had a strong inhibitory effect on lipid peroxidation. We also determined that [Gd@C82(OH)22]n nanoparticles protected cells from oxidative stress in vitro. Using human adenocarcinoma cells (A549 cells) or rat brain capillary endothelial cells (rBCECs), we also demonstrated that [Gd@C82(OH)22]n nanoparticles reduced H2O2-induced ROS formation and mitochondrial damage, measured as reduced mitochondrial dehydrogenase activity and membrane potential. These in vitro results correlate with the previously reported sparing effects of [Gd@C82(OH)22]n nanoparticles on oxidative damage in the livers of tumor-bearing mice. [Gd@C82(OH)22]n nanoparticles efficiently inhibit the growth of malignant MCF-7 solid tumor in vivo. Our overall results suggest that scavenging of ROS plays a role in the potent antitumor effects of [Gd@C82(OH)22]n nanoparticles.
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Materials and Methods |
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; Tang et al., 2005) The nanoparticle characterizations have been described by our group (Chen et al., 2005). In brief, the average size of [Gd@C82(OH)22]n particles in saline solution was 22 nm in diameter, which was measured by high-resolution atomic force microscopy and synchrotron radiation small-angle scattering. Hydrogen peroxide; xanthine; diethylenetriaminepentaacetic acid; DPPH.; 2,2,6,6-tetramethyl-4-piperidone; 5,5-dimethyl-1-pyrroline N-oxide (DMPO); and TiO2 (anatase) were purchased from Sigma-Aldrich (St. Louis, MO). 5-Diethoxyphosphoryl-5-methyl-I-pyrroline N-oxide was supplied by OXIS Research, Inc. (Portland, OR). Xanthine oxidase was obtained from Roche Applied Science (Indianapolis, IN). Bovine liver phosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, AL). 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Bioproducts (Richmond, VA). A Milli-Q water system (Millipore Corporation, Bedford, MA) was used to prepare ultrapure water. All of the other reagents were at least of analytical grade.
DPPH. Scavenging Activity. Following our previously established methodology (Yin et al., 2006), ESR spectroscopy was used to measure scavenging of the DPPH. free radical by [Gd@C82(OH)22]n nanoparticles (Murias et al., 2005). Although DPPH. is a stable, nitrogen-centered radical that has no involvement in physiological processes, attenuation of the ESR signal for DPPH. is one of the methods widely used to demonstrate the ability of a chemical to scavenge reactive oxygen species through donation of a hydrogen atom, or, in some cases, electron transfer (Huang et al., 2005). To measure scavenging of DPPH., the reaction mixture contained 50 µl of [Gd@C82(OH)22]n nanoparticles in 0.85% NaCl and 50 µl of 0.50 mM DPPH. dissolved in ethanol. The final concentration of [Gd@C82(OH)22]n nanoparticles used for measurements was 0.15 mg/ml (100 µM). ESR spectra were recorded at 3, 30, 60, and 120 min after the initiation of the scavenging reaction by adding [Gd@C82(OH)22]n nanoparticles. Spectra were obtained using 20-mW incident microwave power and 100-kHz field modulation of 2 G, at room temperature. The scavenging effect was determined by comparison with a control group lacking [Gd@C82(OH)22]n nanoparticles.
HO. Scavenging Activity. The capacity of [Gd@C82(OH)22]n nanoparticles to scavenge hydroxyl radical was examined by ESR spin-trapping using DMPO as a trapping agent. Hydroxyl radicals were generated by the Fenton reaction, with the reaction mixture containing freshly prepared 0.5 mM DMPO, 0.02 mM FeSO4 (1 mM stock solution, freshly made), and 0.2 mM H2O2. In addition, reaction mixtures contained [Gd@C82(OH)22]n nanoparticles, with final concentrations of 1.67 µM (2.5 µg/ml), 3.34 µM (5 µg/ml), 6.68 µM (10 µg/ml), and 13.34 µM (20 µg/ml), or H2O for the blank. The ESR data were collected at ambient temperature, 6 min after initiating the formation of HO. by addition of FeSO4. The following spectrometer settings were used: microwave power, 10 mW; field modulation frequency, 100 kHz; and modulation amplitude, 1 G.
Inhibition of Lipid Peroxidation. The effects of [Gd@C82(OH)22]n nanoparticles on peroxidation of lipids in liposomes were measured using ESR oximetry. The ESR oximetry measurement is based on bimolecular collision of O2 with a spin probe. Because O2 is paramagnetic, this collision results in spin exchange between O2 and the spin probe, resulting in shorter relaxation times (both T1 and T2). Consequently, ESR signals with broader line widths are observed for the spin probe (Yin et al., 2006). Because the integrated area of the ESR signal over the scanning range is unaffected by these effects on the relaxation times, broadening of the ESR signal of the spin probe is necessarily accompanied by a decrease in the peak height of the ESR signal. In this work, the spin probe 15N-Tempone (PD) (Cambridge Isotope Laboratories, Inc., Andover, MA) was used. Because the line width of this spin probe is very sensitive to O2 concentration, decreasing oxygen concentration as a result of oxygen consumption by lipid peroxidation results in a decreasing line width of 15N-Tempone (PD) spin probe. A time-dependent decrease in line width and increase in intensity of the ESR signal indicates continuous oxygen consumption. By repeated measurement of the spin probe's line widths, one can assess rate of lipid peroxidation in the sample (Scheme 1).
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Liposomes were prepared as described previously (Kusumi et al., 1986). For these experiments, a suspension of 30 mg/ml bovine liver PC liposomes, and 0.1 mM 15N-Tempone (PD) with or without [Gd@C82(OH)22]n nanoparticles (0.1-0.3 mg/ml; 66.8-200.4 µM) was added to a capillary tube. The lipid peroxidation was initiated by adding 25 mM AAPH, which is known to strongly induce lipid peroxidation in cellular and reconstituted membranes (Yin et al., 2006). The capillary tube was then sealed and positioned in the ESR instrument. ESR spectra were recorded at 4-min intervals for 28 min. Signals were obtained with 1-G scanning width, 0.5-mW incident microwave power, and 0.05-G field modulation. All ESR spectra were recorded at the low field line of 15N-Tempone (PD) and at 37°C.
Mitochondrial Dehydrogenase Activity in rBCEC and A549 Cells. The activity of mitochondrial dehydrogenase, a critical enzyme associated with cell survival, was measured according to a modified protocol described previously (Liang et al., 2004). In brief, rBCECs and A549 cells at 90% confluence were cultured with different concentrations of [Gd@C82(OH)22]n nanoparticles for 24 h. The medium was then replaced with fresh medium contained 50 µMH2O2. After treatment with H2O2 for 2 h, cells were washed three times with PBS. The ability of mitochondria to reduce a tetrazolium salt to a formazan dye was used to assess mitochondrial dehydrogenase activity. In brief, a solution of tetrazolium salt (available in the CCK-8 kit; Dojindo Laboratories, Kumamoto, Japan) was added to the culture medium. After incubation for 1.5 h at 37°C, the absorbance at 450 nm was read using a 680 microplate spectrometer (Bio-Rad, Hercules, CA). rBCECs cells were treated with 10 µMH2 O2 to induce oxidative damage. Each group was repeated in triplicate.
Measurement of Mitochondrial Membrane Potential. The fluorescent potentiometric dye JC-1 (Invitrogen, Carlsbad, CA) is a cationic carbocyanine compound that accumulates in mitochondria and can be used to measure mitochondrial membrane potential (
m). When viewed under a fluorescence microscope, JC-1 is seen as a green monomer in the cytosol and as a red aggregate in respiring mitochondria. Mitochondrial damage is measured as a reduction in m (i.e., a decrease in the red/green fluorescence ratio).
A549 or rBCECs cells (1.0 x 105 cells/ml), treated with [Gd@C82(OH)22]n nanoparticles and H2O2 as described above, were labeled with 3 µM JC-1 for 20 min in a 37°C incubator (5% CO2). After cells were washed three times with PBS, the fluorescence was detected using a laser confocal scanning microscope (FV 500; Olympus, Tokyo, Japan). The JC-1 monomer was detected at a 530-nm emission wavelength. The fluorescence of the JC-1 aggregate was measured at 590-nm emission. The cells were also stained with Hoechst 33258 to indicate the nucleus shown in blue. Cells treated with H2O2 were used as the control.
Measurement of Intracellular ROS Concentration. 5-(and 6-)-Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was used to measure intracellular ROS production as reported previously (van Reyk et al., 2001). A549 and rBCECs cells were treated with [Gd@C82(OH)22]n nanoparticles and H2O2 as described above for measurement of mitochondrial dehydrogenase activity. After treatment, the samples were incubated with 5 µM CM-H2DCFDA at 37°C for 30 min in the dark, washed three times with PBS, and then analyzed using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer. The 488-nm excitation wavelength was used to detect the fluorescence intensity of CM-H2DCFDA. Negative controls [without 50 µMH2O2 and without [Gd@C82(OH)22]n nanoparticles] and positive controls [with 50 µMH2O2 and without [Gd@C82(OH)22]n nanoparticles] were included in the treatment groups. Data are representative of three experiments.
Measurement of Malignant Tumor Growth in Tumor-Bearing Mice. Female tumor-bearing nude mice (six/group) were obtained from medical school of Peking University (Beijing, China). At the age of 4 weeks, mice were i.p. administered with [Gd@C82(OH)22]n at the concentration of 2.5 µmol/kg (3.8 mg/ml) in saline once a day consecutively for a period of 14 days. Control animals were injected saline solution only. After animals were sacrificed, the body weight, tumor size, and tumor weight of mice administered with [Gd@C82(OH)22]n or saline solution were determined.
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Results |
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Scavenging of ROS by [Gd@C82(OH)22]n Nanoparticles. To investigate the quenching of physiological ROS by [Gd@C82(OH)22]n nanoparticles, we used the well described classic Fenton reaction involving the reaction of FeSO4 and H2O2 to generate HO. radicals. Upon reaction of the spin trap DMPO with HO., the resulting DMPO-OH adduct showed the typical 1:2:2:1 four-line ESR spectrum (with hyperfine splitting parameter aN = aH = 14.9 G) (Fig. 2). It was observed that [Gd@C82(OH)22]n nanoparticles at as low as 1.67 µM substantially reduced the ESR signal of the DMPO-OH adduct. Scavenging of HO. by [Gd@C82(OH)22]n nanoparticles was concentration-dependent, with the magnitudes of the ESR signal proportionally reduced with the added [Gd@C82(OH)22]n nanoparticles increased from 1.67, 3.34, 6.68, and up to 13.34 µM. It resulted in a greater than 95% reduction in ESR signal for the DMPO-OH adduct when 13.34 µM [Gd@C82(OH)22]n nanoparticles were added (Fig. 2).
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and 1O2, the other two ROS, were also reduced by [Gd@C82(OH)22]n nanoparticles, respectively. The reduction in the ESR signals for 1O2 and was dependent on the concentration of [Gd@C82(OH)22]n nanoparticles (Supplemental Figs. 1 and 2). Inhibition of Lipid Peroxidation in Liposomes by [Gd@C82(OH)22]n Nanoparticles. Reactive oxygen radicals are known to produce a time-dependent peroxidation of the polyunsaturated lipids in plasma membrane. In our study, lipid peroxidation was initiated by AAPH. Inhibition of lipid peroxidation by [Gd@C82(OH)22]n nanoparticles was assessed using ESR oximetry recorded at 4-min intervals for 28 min. The consumption of oxygen-accompanying lipid peroxidation was measured as a time-dependent narrowing of the ESR signal for the spin probe 15N-Tempone (PD). As shown in Fig. 3A, narrowing of the ESR signal was necessarily accompanied by an increase in the peak height of the ESR signal within the scanning range. The results of inhibition of lipid peroxidation by [Gd@C82(OH)22]n nanoparticles at concentrations of 66.8, 133.6, and 200.4 µM are shown in Fig. 3, B-D, respectively; the ESR signal intensities were approximately 80, 75, and 70% of the control (Fig. 3A). All resulted in lower consumption rates of oxygen seen as smaller peak heights in the ESR signal as a result of lower rates of line narrowing of the spin probe. Increasing the concentration of [Gd@C82(OH)22]n nanoparticles from 66.8 to 200.4 µMresulted in an increased inhibition of lipid peroxidation measured as retardation of line narrowing, and an increase in the ESR signal formed by interaction with the spin probe (Fig. 3, B-D). The progressive increases in peak-to-peak signal intensity (and accompanying progressive narrowing of line width) in each panel were due to time-dependent oxygen consumption resulting from lipid peroxidation.
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m. Mitochondrial membrane potential is important for oxidative phosphorylation through electron transfer chain in mitochondria. Dysfunction of mitochondria could enhance the electron leak and ROS generation in cells treated with H2O2. The m, an indicator of mitochondrial damage level, was measured with the JC-1 probe by flow cytometry. In cells untreated with H2O2, the JC-1 probe was seen primarily as red aggregates, indicating substantial uptake by respiring mitochondria (Fig. 4B, A and D). For both rBCEC and A549 cells, treatment with H2O2 resulted in a decrease in red aggregates of the JC-1 probe in mitochondria and an increase in green monomers of the JC-1 probe in the cytoplasm (Fig. 4B, B and E). These results demonstrate H2O2-induced mitochondrial damage and a reduction in m. Pretreatment of cells with [Gd@C82(OH)22]n nanoparticles resulted in partial restoration of mitochondrial uptake of the JC-1, indicating a protective effect against oxidative injury to cellular mitochondria (Fig. 4B, C and F). Protection of mitochondria measured by m is consistent with the measurement of mitochondrial dehydrogenase activity in rBCEC and A549 cells pretreated with [Gd@C82(OH)22]n nanoparticles.
Figure 4C shows the effects [Gd@C82(OH)22]n nanoparticles on the levels of H2O2-induced intracellular ROS. Treatment of rBCEC or A549 cells with H2O2 resulted in a significant increase in intracellular ROS. Pretreatment with [Gd@C82(OH)22]n nanoparticles significantly (P < 0.05) reduced the levels of H2O2-induced ROS in both cell types. The total inhibition percentage in A549 cells was 29.7 ± 2.23% and in rBCECs was 11.9 ± 1.85%, respectively. These results demonstrate a correlation between the bioeffects of [Gd@C82(OH)22]n nanoparticles and their ability to scavenge physiological ROS.
Inhibitory Efficacy of [Gd@C82(OH)22]n Nanoparticles on Tumor Growth in Nude Mice. The growth of human breast carcinomas in mice treated with [Gd@C82(OH)22]n nanoparticles and saline for a period of 14 days was determined. As shown in Fig. 5A, tumor size of mice treated with [Gd@C82(OH)22]n nanoparticles was much smaller than that of control animals. The tumor weight of mice treated with 2.5 µmol/kg [Gd@C82(OH)22]n nanoparticles and with saline is shown in Fig. 5B. The nanoparticle showed significant inhibitory effect 6 days after treatment. In addition, there was less mortality of mice treated with [Gd@C82(OH)22]n nanoparticles than the saline-treated group (data not shown).
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Discussion |
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). [Gd@C82(OH)22]n nanoparticles reduced H2O2-induced ROS formation and mitochondrial damage, measured as reduced mitochondrial dehydrogenase activity and membrane potential in adenocarcinoma cells or rat brain capillary endothelial cells, [Gd@C82(OH)22]n nanoparticles also efficiently inhibit the growth of malignant solid tumors in vivo. The overall data suggest that scavenging of reactive oxygen species by Gd@C82(OH)22 nanoparticles plays a vital role in the inhibition of tumor growth.
Similar to most of chemotherapeutic agents used in clinic, the development of fullerenol nanoparticles for tumor treatment is hampered by their high cytotoxicity. There are modulation factors that can determine whether the developed fullerenol nanoparticles exhibit high therapeutic efficiency and low toxicity (Nishibori et al., 2004). ROS are known to be important factors to initiate the progress of tumor proliferation. In this study, we demonstrate that [Gd@C82(OH)22]n nanoparticles inhibit tumor growth by efficiently scavenging ROS, which promotes tumor proliferation.
With support of our experimental results, we hypothesize that Gd@C82(OH)22 nanoparticles scavenge ROS through electron-electron polarization, forming a semicharge transfer complex with free radicals. The Gd@C82 metallofullerene has aC82 cage and a C2V point group symmetry. The encapsulated gadolinium atom located in the vicinity of the highly conjugated carbon-carbon double bonds of the cage effectively polarizes electron distribution on the carbon cage around the encapsulated gadolinium atom (Nishibori et al., 2004). Our theoretical calculations by an ab initio method (Takabayashi et al., 2004) revealed that the highest occupied molecular orbitals in Gd@C82(OH)22 are localized mainly on the carbon atoms away from the gadolinium atom, whereas almost no frontier orbital distributes on the carbon cage close to the gadolinium atom (Tang et al., 2007a). Thus, encapsulation of the gadolinium transition atom inside of the fullerene cage facilitates polarization of the electrons on the cage, rendering it more accessible for forming the charge-transfer complex with reactive oxygen radicals. Boltalina et al. (1997) reported the electron affinity of several gadolinium metallofullerenes, ranging from C74 up to C82, and determined that in all cases, encapsulization of gadolinium atom inside of the fullerene cage gives rise to an increase in electron affinity (Boltalina et al., 1997). Although the electron affinity of C82 is 3.14 ± 0.06 eV, the electron affinity of Gd@C82 is higher, with a value of 3.3 ± 0.1 eV. Thus, encapsulation of a gadolinium atom inside of the C82 cage enhances the interaction with free radicals through an electron polarization mode.
As shown from our theoretical calculations, besides the effect of the encaged gadolinium atom, the multihydroxyl groups of Gd@C82(OH)22 nanoparticles also induce electron-deficient areas on the cage surface of the molecules, rendering them highly susceptible for attack by the nucleophilic free radicals, such as the hydroxyl radical. The hydroxyl radical polarized toward the Gd@C82(OH)22 cage surface partially loses its electron density, but it can be stabilized (compensated) through forming hydrogen bonding with the proximate hydroxyl proton(s) of Gd@C82(OH)22. Because of its highly electronic deficient and very large cage surface area, the hydroxyl radical-bounded Gd@C82(OH)22 can absorb (trap) another hydroxyl radical through similar electron polarization. The complex species is water-soluble, moves freely within the cells, and is susceptible for interaction with mediums, particularly water, resulting in the chemical formation of hydrogen peroxide, hydroxide, hydrogen molecule, and the recovered intact Gd@C82(OH)22 molecule. The recovered Gd@C82(OH)22 is intact; thus, it can continuously proceed free radical scavenging through the same mechanism. As such, Gd@C82(OH)22 nanoparticles serve as a converter to destroy free radicals into other species but without destroying Gd@C82(OH)22 itself.
We hypothesize that this mechanism to scavenge ROS may be general. Our proposed mechanism suggests that Gd@C82(OH)22 can act as an ROS scavenging sponge, capable of wiping out (scavenging) all types of ROS. Intracellular ROS are known to induce DNA mutation, including the formation of DNA strand breaks and modification DNA bases (Halliwell and Aruoma, 1991; Dizdaroglu, 1992). Therefore, Gd@C82(OH)22 that can scavenge all types of reactive oxygen radicals should possess important biological consequences, including inhibition of tumor cell growth. Investigators are examining the biomedical uses for fullerenes as antioxidants, which can effectively scavenge active radicals or suppress ROS in organ or tissue and reducing oxidative stress caused by tumor progression. Therefore, an increase in antioxidant capacity and ROS scavenging activity would reduce the physiological deterioration and enhance the organism resistance to tumor initiation and promotion. Chemical modifications to alter the surface properties of endohedral metallofullerenol are widely applied to modulate their biological effects, in particular, to reduce or eliminate the nanotoxicity of metallofullerenol in vivo or in vitro (Sayes et al., 2004; Wang et al., 2004). Our study provides new insight in developing novel kinds of nanoparticles as effective chemotherapeutic agents. More research is needed to support the chemotherapeutic mechanism of endohedral metallofullerenes, including [Gd@C82(OH)22]n nanoparticles, to fully exploit them in biomedicine.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ROS, reactive oxygen species; ESR, electron spin resonance; [Gd@C82(OH)22]n nanoparticles, gadolinium endohedral metallofullerenol; HO., hydroxyl radical; , superoxide radical anion; 1O2, singlet oxygen; DPPH, 2,2-diphenyl-1-picrylhydrazyl; rBCEC, rat brain capillary endothelial cell; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; AAPH, 2,2'-Azobis(2-amidinopropane) dihydrochloride; PBS, phosphate-buffered saline; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimida-zolylcarbocyanine iodide; m, mitochondrial membrane potential; CM-H2DCFDA, 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Xing-Jie Liang, Laboratory of Nanobiomedicine and Nanosafety, Division of Nanomedicine and Nanobiology, National Center for Nanoscience and Technology of China, 11, First North Rd., Zhongguancun, Beijing, People's Republic of China, 100190. E-mail: liangxj{at}nanoctr.cn
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bisaglia M, Natalini B, Pellicciari R, Straface E, Malorni W, Monti D, Franceschi C, and Schettini G (2000) C3-Fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis. J Neurochem 74: 1197-1204.[Medline]
Boltalina O, Ioffe IN, Sorokin ID, and Sidorov LN (1997) Electron affinity of some endohedral lanthanide fullerenes. J Phys Chem A 101: 9561-9563.[CrossRef]
Cagle DW, Kennel SJ, Mirzadeh S, Alford JM, and Wilson LJ (1999) In vivo studies of fullerene-based materials using endohedral metallofullerene radiotracers. Proc Natl Acad Sci U S A 96: 5182-5187.
Chen C, Xing G, Wang J, Zhao Y, Li B, Tang J, Jia G, Wang T, Sun J, Xing L, et al. (2005) Multihydroxylated [Gd@C82(OH)22]n nanoparticles: antineoplastic activity of high efficiency and low toxicity. Nano Lett 5: 2050-2057.[CrossRef][Medline]
Chiang LY, Lu FJ, and Lin JT (1995) Free radical scavenging activity of water-soluble fullerenols. J Chem Soc Chem Commun 1: 1283-1284.
Chueh SC, Lai MK, Lee MS, Chiang LY, Ho TI, and Chen SC (1999) Decrease of free radical level in organ perfusate by a novel water-soluble carbon-sixty, hexa(sulfobutyl)fullerenes. Transplant Proc 31: 1976-1977.[CrossRef][Medline]
Dizdaroglu M (1992) Oxidative damage to DNA in mammalian chromatin. Mutat Res 275: 331-342.[CrossRef][Medline]
Dugan LL, Gabrielsen JK, Yu SP, Lin TS, and Choi DW (1996) Buckminsterfullerenol free radical scavengers reduce excitotoxic and apoptotic death of cultured cortical neurons. Neurobiol Dis 3: 129-135.[CrossRef][Medline]
Dugan LL, Turetsky DM, Du C, Lobner D, Wheeler M, Almli CR, Shen CK, Luh TY, Choi DW, and Lin TS (1997) Carboxyfullerenes as neuroprotective agents. Proc Natl Acad Sci U S A 94: 9434-9439.
Halliwell B and Aruoma OI (1991) DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett 281: 9-19.[CrossRef][Medline]
Hsu SC, Wu CC, Luh TY, Chou CK, Han SH, and Lai MZ (1998) Apoptotic signal of Fas is not mediated by ceramide. Blood 91: 2658-2663.
Huang D, Ou B, and Prior RL (2005) The chemistry behind antioxidant capacity assays. J Agric Food Chem 53: 1841-1856.[CrossRef][Medline]
Kusumi A, Subczynski WK, Pasenkiewicz-Gierula M, Hyde JS, and Merkle H (1986) Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase. Biochim Biophys Acta 854: 307-317.[Medline]
Lai HS, Chen WJ, and Chiang LY (2000) Free radical scavenging activity of fullerenol on the ischemia-reperfusion intestine in dogs. World J Surg 24: 450-454.[CrossRef][Medline]
Liang XJ, Shen DW, and Gottesman MM (2004) Down-regulation and altered localization of 
-catenin in cisplatin-resistant adenocarcinoma cells. Mol Pharmacol 65: 1217-1224.
Lin AM, Chyi BY, Wang SD, Yu HH, Kanakamma PP, Luh TY, Chou CK, and Ho LT (1999) Carboxyfullerene prevents iron-induced oxidative stress in rat brain. J Neurochem 72: 1634-1640.[CrossRef][Medline]
Murias M, Jäger W, Handler N, Erker T, Horvath Z, Szekeres T, Nohl H, and Gille L (2005) Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship. Biochem Pharmacol 69: 903-912.[CrossRef][Medline]
Nishibori E, Iwata K, and Sakata M (2004) Anomalous endohedral structure of Gd@C82 metallofullerenes. Phys Rev B 69: 113412.
Sayes CM, Guo W, Lyon D, Byd AM, Ausman KD, Tao YJ, Sitharaman B, Wilson LJ, Hughes JB, West JL, et al. (2004) The differential cytotoxicity of water-soluble fullerenes. Nano Lett 4: 1881-1887.[CrossRef]
Tabata Y, Murakami Y, and Ikada Y (1997) Photodynamic effect of polyethylene glycol-modified fullerene on tumor. Jpn J Cancer Res 88: 1108-1116.[CrossRef]
Takabayashi Y, Rikiishi Y, Hosokawa T, Shibata K, and Kubozono Y (2004) Preferred location of the Dy ion in the minor isomer of Dy@C82 determined by Dy LIII-edge EXAFS. Chem Phys Lett 388: 23-26.[CrossRef]
Tang J, Xing G, Yuan H, Cao WB, Jing L, Gao X, Qu L, Cheng Y, Ye C, Zhao Y, et al. (2005) Tuning electronic properties of metallic atom in bondage to a nanospace. J Phys Chem B 109: 8779-8785.[Medline]
Tang J, Xing G, Zhao F, Yuan H, and Zhao Y (2007a) Modulation of structural and electronic properties of fullerene and metallofullerenes by surface chemical modifications. J Nanosci Nanotechnol 7: 1085-1101.[CrossRef][Medline]
Tang J, Xing J, Zhao Y, Jing L, Yuan H, Zhao F, Gao X, Qian H, Su R, Ibrahim K, et al. (2007b) Switchable semiconductive property of the polyhydroxylated metallofullerene. J Phys Chem B 111: 11929-11934.[Medline]
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, and Telser J (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39: 44-84.[CrossRef][Medline]
van Reyk DM, King NJ, Dinauer MC, and Hunt NH (2001) The intracellular oxidation of 2',7'-dichlorofluorescin in murine T lymphocytes. Free Radic Biol Med 30: 82-88.[CrossRef][Medline]
Wang H, Wang J, Deng X, Sun H, Shi Z, Gu Z, Liu Y, and Zhao Y (2004) Biodistribution of carbon single-wall carbon nanotubes in mice. J Nanosci Nanotechnol 4: 1019-1024.[CrossRef][Medline]
Wang J, Chen C, Li B, Yu H, Zhao Y, Sun J, Li Y, Xing G, Yuan H, Tang J, et al. (2006) Antioxidative function and biodistribution of [Gd@C82(OH)22]n nanoparticles in tumor-bearing mice. Biochem Pharmacol 71: 872-881.[CrossRef][Medline]
Xing G, Zhao YL, Tang J, Zhang B, and Gao XF (2004) Influence of structural properties on stability of fullerenols. J Phys Chem B 108: 11473-11479.
Yin JJ, Kramer JK, Yurawecz MP, Eynard AR, Mossoba MM, and Yu L (2006) Effects of conjugated linoleic acid (CLA) isomers on oxygen diffusion-concentration products in liposomes and phospholipid solutions. J Agric Food Chem 54: 7287-7293.[CrossRef][Medline]
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) compared with saline group (
). ***, P = 0.001, two-tailed Student's t test, nanoparticles versus saline.