Nanoparticles: Synthesis, spectroscopic properties and biological activity

Environmental Health Sciences

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Abstract

We have studied the (photo)toxicity of the fullerene C60 employing four different preparations: (gamma-CyD)2/C60 (gamma-cyclodextrin complexed C60); THF/nC60 (prepared by THF solvent exchange); Son/nC60 (sonication of a toluene solution of C60 mixed with water); and gamma-CyD/nC60 (by heating (gamma-CyD)2/C60 water solution). The absorption spectra indicated that (gamma-CyD)2/C60 in D2O/H2O was present in monomeric state whereas in the other preparations C60 was aggregated. The singlet oxygen quantum yield of (gamma-CyD)2/C60 in D2O was 1.0, the same as C60 dissolved in toluene; emission maximum was 1273 nm. In contrast singlet oxygen phosphorescence generated from the aggregated state of C60 was red-shifted by 10 nm with a very short lifetime in deuterium oxide (2.9 microseconds). While gamma-CyD/nC60 phosphorescence was very sensitive to oxygen it was not quenched by sodium azide. Irradiation (>300 nm) of an aqueous solution of (gamma-CyD)2/C60 in the presence of NADH generated the C60 anion radical which was detected by EPR as a single line (g = 2.0006; p.p. width 1G). When DMPO was present the DMPO-superoxide adduct was observed, confirming the generation of superoxide as a result of the reaction of the C60) anion radical with oxygen. Superoxide was also generated during the irradiation of THF/nC60 and Son/nC60 but only in the presence of NADH. The phototoxicity of the C60 preparations was tested against HaCaT keratinocytes, an immortal human cell line. Sodium azide was more effective than N-acetylcysteine in protecting against (gamma-CyD)2/C60 phototoxicity. The aggregated C60 preparations showed no evidence of (photo)cytotoxicity, with the exception of THF/nC60 which exhibited dark toxicity. The water soluble hydroxylated fullerene fullerol, nano-C60(OH)22-26 has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerols potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic toward HLE B-3 cells maintained in the dark at concentrations higher than 20 uM. Exposure to either UVA or visible light in the presence of >5 uM fullerol induced phototoxic damage. When cells were pretreated with 20 uM lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein alpha crystallin large aggregates of fullerol were produced. Although the acute toxicity of water soluble nano-C60(OH)22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect