Differential biocompatibility of carbon nanotubes and nanodiamonds
Introduction
Carbon nanomaterials are being produced in increasingly larger quantities for many applications due to their novel characteristics, such as enhanced thermal, electronic, mechanical, and biological properties [1]. In biological systems, they have been used as delivery vehicles [2], [3], targeted cancer therapies, tissue scaffolds [4], [5], biosensors, and more [6], [7], [8], [9], [10], [11]. It is envisaged that nanodiamonds (ND) may be particularly well suited for biological applications that require optical transparency, chemical inertness, hardness, and high specific area [6], [12]. Therefore, in view of their biological applications, it is necessary to understand the biocompatibility or toxicity of carbon nanomaterials in either cell-based systems or animal models.
Previous studies in our AFRL laboratory with in vitro cell culture models (macrophages, germ-line stem cells, liver cells, PC-12 cells) have shown that nanoparticles can induce size, composition, and concentration-dependent toxicity [13], [14], [15], [16], [17], [18]. These same factors are likely to influence carbon nanomaterials' biocompatibility or toxicity. Recent studies suggest that the biocompatibility of carbon-based nanomaterials depends strongly on mass, purity, aspect ratio, and surface functional groups. Jia et al., found that low mass and pure fullerenes (C60, > 99.9% purity) were more biocompatible than higher mass and less pure multi-walled carbon nanotubes (MWNT, > 95% purity) or single-walled carbon nanotubes (SWNT, > 90% purity) in guinea pig alveolar macrophages [19]. Magrez et al., found that human lung tumor cell lines were more biocompatible with high aspect ratio MWNTs than compared to carbon nanofibers (CNF) or carbon black (CB) with lower aspect ratios, while acid functionalization increases the toxicity of both CNF and MWNT [20]. The changes in biocompatibility of these carbon nanomaterials, in relation to size or surface chemistry, can be explained by the high density of reactive bonds on carbon black and carbon nanofibers compared to MWNT [20]. One study using relatively large synthetic abrasive diamond powders (100 nm) (that were electron-beam irradiated and annealed for fluorescence, then incubated with kidney cells for 3 h at a concentration of 400 μg/ml) showed very low cytotoxicity for the diamond nanoparticles after they were internalized by the cells [7]. Our work with much smaller 2–10 nm acid or base-purified nanodiamonds at concentrations of up to 100 μg/ml for 24 h shows high biocompatibility in N2A cells [21]. Together these studies suggest that many factors contribute to the biocompatibility of carbon nanotubes while much less is known about the biocompatibility of NDs. Many studies have examined the biocompatibility of diamond surfaces [22], [23], but simple extrapolation of surface biocompatibility data to diamond nanoparticles in solution has been shown to be impossible [9], [24].
Other studies that have used in vitro cell culture models focused on lung or skin cells due to the risk of exposure in occupational or commercial settings [25], [26], [27]. However, it is unclear whether these nanomaterials can reach the nerves associated with these organs either through internalization through the skin and contact with olfactory nerves or translocation across the blood-brain barrier. In the present study, we examined both neuronal (neuroblastoma) and lung (alveolar macrophage) cell lines for biocompatibility in aqueous suspensions of carbon nanomaterials (e.g. ND, SWNT, MWNT, CB) at concentrations ranging from 25–100 μg/ml for 24 h. We further examined the morphological and subcellular effects of these nanomaterials on mitochondrial membrane permeability and reactive oxygen species (ROS) generation.
Section snippets
Nanomaterials characterization
Multi-walled carbon nanotubes (MWNTs) were purchased from Tsinghua University, Beijing, China while single-walled nanotubes (SWNTs) were received from Rice University. Nano-sized carbon black was from Cabot (CB) and micron-sized cadmium oxide (CdO) was from the Fluka Chemical Company. Nanodiamonds (NDs) were generously supplied by NanoCarbon Research Institute Ltd. in Japan and were synthesized according to previously reported detonation techniques [28], [29]. Nanomaterials were UV-sterilized,
Nanomaterials characterization
The wide range of primary sizes and shapes of carbon nanomaterials were investigated with transmission electron microscopy (Fig. 1A–D). Individual cubic nanodiamonds (NDs) with sizes ranging from 2–10 nm (Fig. 1A) were smaller than more spherical fine carbon black (CB) nanoparticles (Fig. 1B) with sizes of approximately 20 nm. Single-walled carbon nanotubes (SWNTs) existed in bundles with diameters up to 25 nm and lengths over 3 μm (Fig. 1C), while multi-walled carbon nanotubes (MWNTs) had
Conclusions
In the present study, we found that NDs were more biocompatible than CB, MWNTs, or SWNTs, respectively, in two different cell types (neuroblastoma and alveolar macrophage), though macrophages are more sensitive to the carbon nanomaterials likely due to their innate response to foreign materials. Examination of the cell morphologies revealed that neuroblastoma cells can lose their neurite extensions after incubation with carbon nanomaterials at high (100 μg/ml) concentrations, whereas
Acknowledgments
The authors thank Col J. Riddle of AFRL/HEPB for his strong support and encouragement for this research and the Nanoscale Engineering Science & Technology (NEST) Laboratory at the University of Dayton for use of TEM. Ms. Amanda Schrand is funded by the Biosciences and Protection Division, Air Force Research Laboratory under the Oak Ridge Institute for Science and Education, Oak Ridge TN. Ms. Schrand is also funded by the Dayton Area Graduate Studies Institute (DAGSI). L.D. and E.O. thank NEDO
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