Hydroxylation of multi-walled carbon nanotubes: Enhanced biocompatibility through reduction of oxidative stress initiated cell membrane damage, cell cycle arrestment and extrinsic apoptotic pathway

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Highlights

  • MWCNTs-OH induced significantly less oxidative stress compared with p-MWCNTs.

  • MWCNTs-OH lead to less cell membrane damage compared with p-MWCNTs.

  • MWCNTs-OH decreased arrestment of cell cycle at G0/G1 than p-MWCNTs.

  • MWCNTs-OH exhibited significantly lower tendency to activate caspase-8 than p-MWCNTs.

Abstract

Modification of CNTs with hydroxyl group promotes their applications in biomedical area. However, the impact of hydroxylation on their biocompatibility is far from being completely understood. In this study, we carried out a comprehensive evaluation of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) on the human normal liver L02 cell line, and compared it with that of pristine multi-walled carbon nanotubes (p-MWCNTs). Results demonstrated that compared with p-MWCNTs, MWCNTs-OH induced significantly lower oxidative stress as indicated by the level of intracellular antioxidant glutathione (GSH), subsequently lead to less cell membrane damage as demonstrated by lactate dehydrogenase (LDH) leakage assay, and showed slightly decreased arrestment of cell cycle distribution at G0/G1. More interestingly, MWCNTs-OH exhibited significantly lower tendency to activate caspase-8, a key molecule involved in the extrinsic apoptotic pathway. All these in vitro results demonstrated that hydroxylation of MWCNTs enhanced their biocompatibility compare with p-MWCNTs.

Introduction

Carbon nanotubes (CNTs), which are high-aspect-ratio nanomaterials, can be classified into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) (Ajayan, 1999). CNTs have attracted increasing attention because of their unique chemical and physical properties and have shown diverse potential applications in industrial and medical fields (Li et al., 2010). However, before CNTs entering into the real clinical usage, the biological toxicology of these nanomaterials must be addressed (Du et al., 2013). According to the recent studies, many aspects can impact the biocompatibility of CNTs, such as length (Cheng and Cheng, 2012), thickness (Fenoglio et al., 2012), aggregation (Xue et al., 2016), and metallic content (Aldieri et al., 2013). For biomedical applications of CNTs, enhanced water dispersibility of these nanomaterials is needed. There are many strategies to increase the hydrophilicity of CNTs. Surface modification of CNTs with hydroxyl groups is one of the most used methods. And CNTs can further conjugate with other molecules after hydroxylation. Here, we were interested in understanding whether the biocompatibility can be improved under such modification, which would have great significance in the biomedical application of these nanomaterials. However, to our best knowledge, the impact of hydroxylation of CNTs on their biocompatibility has far from being thoroughly investigated.

Although some other studies have been carried out to investigate the bio-effects of functionalized CNTs, however, the interaction of hydroxylated CNTs with biological system has seldom been studied and the underlying mechanisms are poorly understood, and there are many divergent findings. Some researchers found that oxidation modified CNTs own enhanced biocompatibility. Dumortier et al. found that oxidation/amidation modified CNTs at low concentration have non-cytotoxicity to primary immune cells and preserved the functionality of the cells (Dumortier et al., 2006). Sayes et al. reported that phenyl-SO3H and phenyl-(COOH)2 modification enhanced the biocompability of SWCNTs on human dermal fibroblasts, and the biocompatibility enhanced with increased intensity of functionalization (Sayes et al., 2006). MWCNTs-COOH attenuated the cellular toxicity to D384 cells and A549 cells at doses of 1–100 μg/mL compared to p-MWCNTs (Coccini et al., 2010). MWCNTs-COOH significantly reduced the extent of pulmonary fibrosis with decreased generation of pro-fibrogenic cytokines and growth factors compared with that of p-MWCNTs (Li et al., 2013). Ursini et al. reported that MWCNTs-OH at concentrations of 1–20 μg/mL showed less cytotoxicity compared with p-MWCNTs (Ursini et al., 2012). Recently, an in vivo study indicated that MWCNTs-OH induced significant lower inflammatory response than p-MWCNTs when used as scaffolds for bone regeneration (Mikael et al., 2014). MWCNTs-OH almost caused no lethal effect to Daphnia magna at concentration of 0.01, 0.10, 0.50 or 1.00 mg/L (Heffron et al., 2014). Although these researches have been carried out, more fundamental mechanisms are still need to be revealed.

On the contradictory, enhanced cytotoxicity of functionalized CNTs compared with p-MWCNTs has shown. It was reported that acid-treated SWCNTs exhibited higher toxicity to human endothelial cells (Gutierrez-Praena et al., 2011). Similarly, an increase in cytotoxicity and inflammatory response in RAW 264.7 macrophages was observed when p-MWCNTs were functionalized with carboxyl or polyethylene glycol (PEG) groups (Zhang et al., 2012).

Multiple mechanisms might be involved in the biological effects (Dong and Ma, 2015). In our previous study (Liu et al., 2014), we found that hydroxylation improved the biocompatibility of MWCNTs through limiting the mitochondria mediated apoptotic pathway. Here, we report a more comparative study on the effects of MWCNTs-OH and p-MWCNTs on L02 cells. The methodology used in this study including the investigation of the oxidative stress, the subsequent cell membrane integrity damage, the cell cycle arrestment and the activation of extrinsic apoptotic pathway to obtain a comprehensive understanding of the fundamental biological mechanisms of the hydroxylated MWCNTs compared with p-MWCNTs and give more references for the application of hydroxyl functionalized MWCNTs.

Section snippets

Materials

MWCNTs-OH and p-MWCNTs (10∼30 μm in length and 10∼20 nm in diameter) were obtained from Chengdu Organic Chemicals Co., Chinese Academy of Sciences (Chengdu, China). The MWCNTs were synthesized via the carbon vapor deposition (CVD) method. The purity of MWCNTs was over 95% and surface hydroxyl group modification of MWCNTs-OH was 2.00 wt%. RPMI-1640 cell media (Cat. No. SH30027.01) was purchased from Hyclone (Logan, Utah, USA). L02 cells (third passage) were purchased from American Type Culture

Characterization of MWCNTs

The transmission electron microscope (TEM) images of the dispersed MWCNTs confirmed that the average diameter of p-MWCNTs and MWCNTs-OH was 10–20 nm (Fig. 1). The morphologies and size of both MWCNTs were also confirmed in our previous study with scanning electron microscope (SEM) (Liu et al., 2014), which showed that the MWCNTs owned tubular structure and the length ranged from 10 to 30 μm. The surface presence of hydroxyl groups was characterized by X-ray photoelectron analysis, which showed

Conclusions

The results in this research showed that exposure to MWCNTs-OH and p-MWCNTs initiated oxidative stress and subsequently caused cellular membrane damage, cell arrestment at G0/G1 phase, as well as the activation of extrinsic apoptotic pathway. Compared to p-MWCNTs, MWCNTs-OH induced a significant attenuation of these phenomena. This study contributes to more understanding of the mechanisms of enhanced biocompatibility of hydroxylated MWCNTs.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81301258), Hunan Provincial Natural Science Foundation of China (2016JJ2161), Specialized Research Fund for the Doctoral Program of Higher Education of China (20130162120078), China Postdoctoral Science Foundation (2013M540644), International Postdoctoral Exchange Fellowship Program (2014) 29 (20140014) and Shenghua Yuying Project of Central South University.

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