Elsevier

Biomaterials

Volume 26, Issue 36, December 2005, Pages 7587-7595
Biomaterials

Nano-C60 cytotoxicity is due to lipid peroxidation

https://doi.org/10.1016/j.biomaterials.2005.05.027Get rights and content

Abstract

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses ⩾50 ppb (LC50=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.

Introduction

Water-soluble fullerene systems are promising candidates for many medical technologies and have been proposed as crucial components for emerging electronic, optical and mechanical materials [1], [2]. Given the widespread applications and their impending commercialization, both humans and environmental systems will be increasingly exposed to materials like C60 in the near future; thus, early evaluations of their health effects are valuable [3]. Previously, we have evaluated the differential cytotoxicity of a series of water-soluble fullerene species and concluded that changes in the fullerene cage structure had substantial impact on in vitro cytotoxicity [4]. As the number of hydroxyl or carboxyl groups on the surface of the fullerene cage was increased, cytotoxicity decreased over seven orders of magnitude. The series of water-soluble fullerenes included nano-C60, tris-malonic acid-C60 (or C3), Na+2−3[C60O7−9(OH)12−15](2−3)−, and C60(OH)24. Using two separate methods, we determined that the nano-C60 generated substantially more reactive oxygen species (ROS) than the other species under cell-free conditions. The ROS generation monotonically decreased with increasing derivatization of the fullerene cage. The dramatic cytotoxicity observed for nano-C60 warranted further evaluation as presented here. Additional studies were also conducted to probe the mechanisms governing the cytotoxicity of nano-C60 and confirm the importance of oxidation.

Water-soluble fullerene derivatives are most commonly formed by deliberate synthetic methodologies and typically have altered cage chemistry and high (>100 ppm) water solubility. In contrast, the nano-C60 colloid investigated here is only sparingly soluble; it is produced by the addition of organic soluble C60 to water [5], [6], [7], [8]. The same substance is also found when solid C60 is stirred in tap water for 2 months [9]. Because of its ease of formation, and stability in water, nano-C60 is likely to be an important form of C60 in natural aqueous systems.

The full characterization of the nano-C60 water suspension is published in two separate reports [4], [10]. Proof of the presence of C60 in the aqueous suspension include electron diffraction via cryo-TEM, chromatographic profiles and signature spectroscopic peaks identify the presence of C60 in the aqueous solution. In addition, microscopy images show the size, shape and crystallinity of the C60 colloids. While the method of nano-C60 preparation may leave intercalated THF, mass spectroscopy and liquid chromatography prove that more than 90% by weight of the suspension is C60, i.e. <10% of the suspension is residual solvent. Viability controls confirm that this residual solvent does not contribute to cell death or generation of ROS. The structural of the nano-C60 colloid consists of a pristine C60 core (of 10–1000 C60 molecules, depending on size of crystal) surrounded by a low derivatized C60 layer that forces the aggregate to be miscible in the aqueous phase. The extent of the derivatization is 3 groups or less, composed of either hydroxylated or oxidized fullerenes (confirmed by nuclear magnetic resonance and Fourier transform infrared spectroscopy). The negative surface charge of the aggregate is further evidence of a hydrophilic surface derivation. Because of this low degree of derivatization, we cannot fully identify the exact chemical composition of these groups.

Previous reports by Oberdorster suggest the brain and liver of largemouth bass produce changes in glutathione production once exposed to nano-C60. Therefore, we hypothesize that the human cell lines hDF, Human liver carcinoma cells (HepG2), and NHA might be affected by nano-C60. More specifically, within these cell lines, we concentrated on examining the effect of nano-C60 on the membrane of the cell, since we previously reported that nano-C60 produces oxygen radicals in water.

Section snippets

Materials and methods

All chemicals were purchased through Sigma Aldrich at highest purity unless otherwise stated and experiments were performed minimally in triplicate. Data are presented as mean ± standard deviation, and a student's t-test was used to determine significance.

Results

Cell viability was determined following 48 h exposure to fullerenes. We previously found that LDH release does not occur at 1, 12, or 30 h after inoculation with the nano-C60. For this reason, all LDH measurements were performed after 48 h. The LC50 values, determined from the dose response curves shown in Fig. 1, are as follows: for the HDF cells, 20 ppb; for HepG2, 50 ppb; and for NHA cells, 2 ppb. Since substantial cytotoxicity was observed for nano-C60, the mechanisms by which nano-C60 damages

Discussion

We investigated the effect of water-soluble fullerene aggregates, nano-C60, on HDF, HepG2, and NHA cells in culture. Nano-C60 demonstrated significant toxicity in previous cell culture studies, while a highly hydroxylated, water-soluble fullerene, C60(OH)24 did not [4]. In these studies, we have determined that lipid peroxidation and resultant membrane damage are responsible for the cytotoxicity of nano-C60. In addition, the oxidative damage and toxicity of nano-C60 were prevented by addition

Conclusion

The response of a cell to a nanomaterial can aid in the evaluation of the material for medical applications and environmental fate. Given the enormous range of nanoparticle types, morphologies and surface chemistries, as well as the uncertain form of nanoparticles in future applications, toxicological testing that only provides a measure of hazard is not useful. Instead, toxicology in this emerging area must provide a basis for predicting systematically how a nanoparticle's biological behavior

Acknowledgements

We thank Marcella Estrella for technical assistance with the cell cultures; Prof. Jane Grande-Allen for instrument use; and John D. Fortner, Delina Lyon, and Adina M. Boyd for supplying the nano-C60 sample. This work was financially supported by the Center for Biological and Environmental Nanotechnology (NSF EEC-0118007).

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