Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2

Abstract

BackgroundGraphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets.MethodsGraphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy.ResultsSupplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 μg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity.ConclusionsGO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 μg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications.

Frameworks and tools for risk assessment of manufactured nanomaterials

“…Advanced REACH Tool (ART) (Fransman et al, 2009) Occupational exposure assessment (Gorman et al, 2012) Occupational/consumer exposure assessment (Gottschalk et al, 2010a, 2010b, Sun et al, 2014, Gottschalk et al, 2009, Gottschalk et al, 2011 (Yokel et al, 2009, Yokel et al, 2012, Hardas et al, 2010, Dan et al, 2011 In vivoIkinetic tools for human exposure and hazard assessment (Lammel et al, 2013, Lammel andNavas, 2014) In vitro and in vivo tools for human hazard assessment…”

“…cell membranes, metabolism and clearance mechanisms (Lammel et al, 2013). These tools aim to prioritize MNs for further testing and to determine realistic exposure concentrations to be applied in in vivo testing.…”

Quercetin-mediated synthesis of graphene oxide&ndash;silver nanoparticle nanocomposites: a suitable alternative nanotherapy for neuroblastoma

“…65,77 For instance, mitochondrial depolarization is associated with mitochondrial dysfunction and the overproduction of ROS. 68,78 Recently, Mari et al 30 reported that GO nanoribbons induced toxicity in human neuroblastoma cancer cell lines such as SK-N-BE(2) and SH-SY5Y. Altogether, the combination of GO and AgNPs in a single platform such as GO-AgNPs could modulate the regulation of mitochondria and increase the generation of ROS.…”

“…67 Other studies showed that exposing cells to GO results in the perturbation of the mitochondrial structure and function, as characterized by a decrease in the MMP and the dysregulation of mitochondrial Ca 2+ homeostasis. 68,69 Similarly, nano-sized GO, pristine graphene, and graphene quantum dots caused time-and concentration-dependent decreases in the MMP. 70,71 Altogether, the data from our findings clearly indicate that GO-AgNPs could cause mitochondrial dysfunction through loss of MMP.…”

The potentiation effect makes the difference: Non-toxic concentrations of ZnO nanoparticles enhance Cu nanoparticle toxicity in vitro

“…Ultrathin sections of cells were analyzed by TEM to reveal the intracellular accumulation/distribution of NPs and possible ultrastructural modifications. The method used has been described in Lammel et al (2013), which is briefly shown in the Supplementary Materials. A JEOL 1010 JEM (JEOL) microscope operating at an acceleration voltage of 100 kV with integrated energy dispersive X-ray (EDX) spectroscopy (Oxford INCA) was used.…”