Elsevier

Toxicology in Vitro

Volume 41, June 2017, Pages 205-213
Toxicology in Vitro

Comparative in vitro study of single and four layer graphene oxide nanoflakes — Cytotoxicity and cellular uptake

https://doi.org/10.1016/j.tiv.2017.03.005Get rights and content

Highlights

  • The in vitro study confirmed comparable cytotoxicity of 1- and 4-layer GO.

  • PEGylation improved dispersibility of the both GO nanoflakes.

  • Stronger inhibition of cell proliferation by 1-layer GO-PEG was detected.

  • Markedly higher ROS generation by 1-layer GO-PEG was observed.

  • Cell imaging revealed efficient internalization of the both GO nanoflakes.

Abstract

In recent years, graphene and its derivatives have been extensively investigated because of their unique properties, which can be used in many fields including biomedical applications. Therefore, detailed biological study is required. In the current paper the detailed toxicological studies on single and four layer graphene oxide (GO) nanoflakes is presented. The morphology and size of the nanomaterials were characterized via atomic force microscopy. Cytotoxicity, proliferation and internalization study were performed using various methods, including optical, confocal and Raman microscopy imaging, flow cytometry analysis, colorimetric and luminescent cell assays. Our first findings undeniably show that the nanomaterials' functionalization has a considerable impact on their behavior in a biological environment. The cytotoxicity assay confirmed comparable, dose dependent cytotoxicity of single and four layers GO flakes. The differences between these two nanomaterials became more distinct during cell proliferation study and ROS detection. Namely, markedly stronger inhibition of cell proliferation and higher ROS generation by one-layer GO-PEG than four-layer GO-PEG were observed. Cell imaging revealed efficient internalization of the both GO nanoflakes in a time dependent manner. These findings emphasize the role of number of layer and functionalization in GO toxicological characteristics and may provide helpful information for their further biomedical applications.

Introduction

Graphene is a part of a bigger class that has been named as graphene family nanomaterials (GFNs) that consist of carbon structures with different chemico-physical properties and different number of layers. GFNs possess extraordinary electronic, optical, thermal and mechanical properties, which can be used in many fields (Sanchez et al., 2012). Moreover, high specific area and facile functionalization make them promising materials for biomedical applications, ranging from drug/gene delivery, biological sensing and imaging, antibacterial materials, scaffold for cells' culture and tissue engineering (Shen et al., 2012; Chung et al., 2013).

The biocompatibility of graphene-based nanomaterials is not only dose and time dependent but also may be connected with physical parameters, number of layers, size, shape, surface functionalization and synthesis (Chatterjee et al., 2015). Therefore, new materials in biomedical applications require detailed studies. Many authors focused on graphene oxide (GO) that demonstrates considerable advantages over other members of GFNs (Chatterjee et al., 2015). In the current paper the biological characteristics of single and four layer GO nanoflakes of similar length is presented. The number of GFNs layers is an important parameter relevant for their biological effects, as it determines specific surface area, bending stiffness and adsorptive capacity. Namely, specific area is inversely proportional to number of layers, thus adsorptive capacity for biological molecules increase significantly as layer number decreases. In contrary, bending stiffness increases with the number of layers (Sanchez et al., 2012, Chatterjee et al., 2015).

Prevention of aggregation is an important factor for graphene sheets because most of their unique properties are only connected with the single layer. Aggregation can be reduced by the attachment of surfactant or by controlled chemical conversion of GO colloids (Li et al., 2008, Wang et al., 2013). Many researchers confirmed that PEGylation (functionalization with polyethylene glycol) made nanomaterials much more dispersible and stable in different solutions, even rich in salts or proteins, such as cell media and serum (Wang et al., 2013; Sun et al., 2008). Moreover functionalization can profoundly change their cytotoxicity by attenuating the hydrophobic interactions between graphene or GO with cells and tissues (Zhang et al., 2012). Therefore, in the first step the biocompatibility of the GO nanoflakes in pristine form and after PEGylation was investigated in the present study. Next, more detailed research on less cytotoxic PEGylated nanostructures was continued. Biocompatibility and internalization study were performed using various methods, including optical and confocal microscopy imaging, flow cytometry analysis, colorimetric and luminescent cell assays.

Section snippets

Nanomaterials synthesis and functionalization

The graphene oxides (GO) synthesis routes were reported previously (Marcano et al., 2010; Wojtoniszak and Mijowska, 2012). The 4-layer GO synthesis proceeded as follows, 1 g of graphite was dispersed in a mixture of perchloric and nitric acids (350 mL, 4:3—volume ratio), and next potassium chromate (6 g) was added. The mixture was then heated to 50 °C for 24 h. The obtained mixture was centrifuged and washed three times with ethanol (200 mL) and 10% hydrochloric acid (200 mL) to remove residual metal

Characterization of nanomaterials

The thickness of the nanostructures was estimated using atomic force microscopy. The thickness of the 1-layer and 4-layer GO flakes is about 1.0 nm and 3.0 nm, which corresponds to one and four layer graphene (Fig. 1). Detailed chemical and physical characterization of the graphene oxide is described in the previous publication (Wojtoniszak and Mijowska, 2012). The length distribution of the GO flakes was located between 1 and 25 μm. Mean length for the 1-layer and 4-layer GO ranged from 1 to 15 

Discussion

Herein, we have shown the in vitro study on the cytotoxicity of graphene oxide (GO) nanoflakes. The cell morphology, viability, membrane integrity and ROS level in MCF7 cells were evaluated after GO exposure. We estimated the influence of the number of graphene layers and PEG functionalization on GO cytotoxicity. The MCF7 cells used in the present study are commonly applied in the nanomaterial cytotoxicity and drug delivery assays (Hu et al., 2012, Yue et al., 2012).

The microscopy imaging

Conclusions

The aim of the current study was to evaluate and compare the cytotoxicity of two types of GO nanoflakes with similar length distribution and different number of layers. Current findings confirmed that PEGylation has a considerable impact on nanomaterial behavior in a biological environment and their interactions with MCF7 cells. Namely, functionalization enhanced nanomaterials' dispersibility and stability in cell culture medium, preventing nanomaterial agglomeration and sedimentation as well

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Acknowledgments and declaration of interest statement

The authors have no financial involvement with any organization or entity with a financial interest in or financial c with the subject matter or materials discussed in the manuscript.

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