Graphene Oxide Nanosheets Disrupt Lipid Composition, Ca²⁺ Homeostasis, and Synaptic Transmission in Primary Cortical Neurons

Abstract

Graphene has the potential to make a very significant impact on society, with important applications in the biomedical field. The possibility to engineer graphene-based medical devices at the neuronal interface is of particular interest, making it imperative to determine the biocompatibility of graphene materials with neuronal cells. Here we conducted a comprehensive analysis of the effects of chronic and acute exposure of rat primary cortical neurons to few-layer pristine graphene (GR) and monolayer graphene oxide (GO) flakes. By combining a range of cell biology, microscopy, electrophysiology, and "omics" approaches we characterized the graphene-neuron interaction from the first steps of membrane contact and internalization to the long-term effects on cell viability, synaptic transmission, and cell metabolism. GR/GO flakes are found in contact with the neuronal membrane, free in the cytoplasm, and internalized through the endolysosomal pathway, with no significant impact on neuron viability. However, GO exposure selectively caused the inhibition of excitatory transmission, paralleled by a reduction in the number of excitatory synaptic contacts, and a concomitant enhancement of the inhibitory activity. This was accompanied by induction of autophagy, altered Ca(2+) dynamics, and a downregulation of some of the main players in the regulation of Ca(2+) homeostasis in both excitatory and inhibitory neurons. Our results show that, although graphene exposure does not impact neuron viability, it does nevertheless have important effects on neuronal transmission and network functionality, thus warranting caution when planning to employ this material for neurobiological applications.

Biotransformation and Biological Interaction of Graphene and Graphene Oxide during Simulated Oral Ingestion

“…Representative flakes of FLG and GO, with many visible wrinkles are shown in Figure a,b, respectively. The statistical analysis of TEM images revealed broad lateral size distributions of both GRMs in water dispersion (Figure c,d), in line with previously reported results . The mean flake length was similar for both materials and around 400 nm.…”

“…After exfoliation, melamine was removed by filtration with hot water to obtain stable dispersions of FLG. The final FLG concentration was estimated to be 0.09 mg mL −1 with melamine traces (0.09 ppm) as reported elsewhere . GO flakes were obtained through oxidation of carbon fibers (GANF Helical‐Ribbon Carbon Nanofibers, GANF).…”

In Vitro Evaluation of Biocompatibility of Uncoated Thermally Reduced Graphene and Carbon Nanotube-Loaded PVDF Membranes with Adult Neural Stem Cell-Derived Neurons and Glia

“…In spite of these potential applications, other studies have reported that GBNs can cause cytotoxic and genotoxic effects on cell lines (PC12, neuroblastoma, and A549 cells), mesenchymal stem cells (Zhang et al, 2010; Chang et al, 2011b; Akhavan et al, 2012; Lv et al, 2012; Bianco, 2013; Tu et al, 2013b), and neurons (Bramini et al, 2016). CNTs, particularly if used as produced materials, can also induce toxic effects on neural cells in part due to the presence of CNT aggregates, impurities such as amorphous carbon and metallic nanoparticles (Jakubek et al, 2009; Cellot et al, 2010; Wu et al, 2012; Chen et al, 2013; Meng et al, 2013; Bussy et al, 2015).…”

Carboxylated graphene oxide promoted axonal guidance growth by activating Netrin‐1/deleted in colorectal cancer signaling in rat primary cultured cortical neurons

“…It maybe relate to the unique structural properties of GO and GOOH such as enriched oxygen‐containing groups and great biocompatibility . While previous study indicated that GO‐COOH has not side effect on neuron viability, thus graphene application can be considered for neural regeneration.…”