Real-time electrical impedance analysis of graphene oxide cytotoxicity in human epithelial lung cells

Graphene oxide (GO) is a promising two-dimensional nanomaterial (NM) due to its unique physicochemical properties, yet concerns persist regarding its potential cytotoxicity. This study aimed to evaluate the cytotoxic response of GO in human alveolar epithelial A549 cells using real-time cell analysis (RTCA), a label-free impedance-based method, complemented by the classical MTT assay and supported by detailed physicochemical characterization and ISO-aligned measurement controls.

GO was characterized by UV-Vis Spectroscopy, Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Raman Spectroscopy, and X-Ray Photoelectron Spectroscopy (XPS) following ISO/TR 13014:2012 guidance. Human alveolar epithelial A549 cells were exposed to GO (1-100 µg/mL) and monitored by RTCA, a non-invasive, label-free impedance-based assay. In parallel, cell viability was evaluated by the MTT assay after 72 h of exposure. To confirm assay robustness, murine L-929 fibroblasts-recommended by ISO 10993-5 for cytotoxicity screening-were included as a reference model. Triton X-100 served as a positive control.

GO did not induce relevant changes in cell index or mitochondrial activity up to 100 µg/mL. Both A549 and L-929 cells maintained viability above the 70% ISO threshold, with consistent results across independent experiments. The complementary behavior of RTCA and MTT confirmed the reliability of the results.

The GO synthesized in this work showed no detectable cytotoxic or cytostatic effects in either cell line under the tested conditions. The ISO-aligned framework, combining label-free and endpoint assays, provided a structured basis for interpreting these results and for further evaluation in more complex models.