Live Imaging of Label-Free Graphene Oxide Reveals Critical Factors Causing Oxidative-Stress-Mediated Cellular Responses

Abstract

The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and the pulmonary system. The aim of the present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions, s-GO (50-200 nm) and l-GO (5-15 μm), were produced and thoroughly characterized. Exploiting intrinsic fluorescence of graphene oxide (GO) and using confocal live-cell imaging, the behavior of the cells in response to the material was visualized in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated reactive oxygen species (ROS) levels, pro-inflammatory response, and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, the lateral dimension of GO played a more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO-induced cytotoxicity.

Corrosion Motivated ROS Generation Helps Endow Titanium with Broad‐Spectrum Antibacterial Abilities

“…Recent research found that OM is susceptible to reactive oxygen species (ROS), and the Gram‐ bacteria is easier to be killed in an oxidative stress environment . Although various ROS producing agents are available, such as graphene and its derivatives, iron nanoparticles, noble metal, or doping titanium oxides, it remains a challenge to design a broad‐spectrum antibacterial surface fulfilling the various requirements of titanium implants. For instance, the developed surface must be biocompatible.…”

Graphene Oxide as 2D Platform for Complexation and Intracellular Delivery of siRNA

“…These vesicles sequestered the material for more than 24 h in the apical region of the cell, with no apparent trafficking taking place towards endosome-lysosomal or extracellular compartments ( Figure S8, Video S3). The same pattern of uptake was found in other non-phagocytic cell lines when GO flakes of similar lateral dimensions were also dispersed in serum-free cell culture medium for the first 4 h 19 . In other studies, GO has been reported to enter cells mainly via endocytosis, although fewlayer GO flakes of lateral dimensions under 10 µm can potentially pierce the cell membrane directly 38 .…”

“…Next, we followed the internalization of GO and siRNA in MEFs. Thanks to the intrinsic fluorescence of the material, GO uptake can be monitored by confocal microscopy without further functionalization or labelling, as demonstrated in a previous study from our laboratory 19 . However, fluorophore quenching impeded the visualization of a fluorescently-labelled siRNA complexed onto GO (data not shown).…”

“…We first examined any cytotoxic responses to GO:siRNA complexes on a primary mouse cell culture system. A GO material similar to the one utilized herediffering only in the graphite source, but with practically identical physicochemical propertiesdid not induce cytotoxicity in a human lung epithelial cell line (BEAS-2B) at the doses included in this study 19 . Here, mouse embryonic fibroblasts (MEFs) obtained from CD1 mice were exposed to GO:siRNA complexes, where siRNA was a scrambled sequence known to be inert to cells.…”

Biocompatibility Considerations in the Design of Graphene Biomedical Materials

“…Among all material properties that can be ascribed for GBM adverse effects, as previously reviewed in details, lateral dimensions, surface chemistry and reactivity, thickness, agglomeration, and the presence or absence of biomolecule corona seem to be the most essential toxicity determinants for GBM nanoforms ( Figure ).…”