Angiotensin II-Induced Hypertrophy in H9c2 Cells Reveals Severe Cytotoxicity of Graphene Oxide

This study investigates the differential cytotoxicity of reduced graphene oxide (RGO) and graphene oxide (GO) particles using an angiotensin II (Ang II)-induced hypertrophy model in H9c2 cells. Herein, GO particles were synthesized from graphite, and subsequent reduction was carried out to obtain RGO particles. To ensure a thorough assessment of particle size, functionalization, and purity, the particles were characterized by using UV-vis absorbance spectroscopy, dynamic light scattering, X-ray photoelectron spectroscopy, FTIR spectroscopy, Raman spectroscopy, and scanning electron microscopy. Comprehensive characterization revealed that the transformation from GO (∼21.6% content of oxygen) to RGO (∼13.3% content of oxygen) results in an enrichment in the proportion of sp2 carbon. Additionally, rat cardiac myoblasts of the H9c2 cell line were subjected to Ang II to induce cellular hypertrophy, leading to cytoskeleton remodeling, increased cardiac myocyte surface area, extracellular matrix alterations, and collagen type 1a upregulation. To evaluate cytotoxicity, H9c2 cells were treated with RGO and GO suspensions at concentrations ranging from 1 to 10,000 μg/mL, and metabolic viability was assessed in both concentration- and time-dependent assays. GO and RGO reduced the viability of H9c2 cells; however, the metabolic viability assays showed that the half-maximal inhibitory concentration (IC₅₀) values for GO and RGO were significantly lower in hypertrophic cardiomyocytes, with GO exhibiting an IC₅₀ of 12.6 ± 10.7 μg/mL and RGO exhibiting an IC₅₀ of 86.3 ± 12.9 μg/mL, compared to control cells (676.0 ± 80.3 μg/mL for GO and 152.9 ± 40.1 μg/mL for RGO). These results demonstrate that under hypertrophic conditions, there is a significant increase of cytotoxicity for GO (50-fold increase) in comparison to RGO (1.7-fold increase). It was demonstrated that GO particles create a pro-oxidative environment that ultimately leads to mechanistic impairments and cell death. Vulnerable populations predisposed to cardiac damage may be at increased risk of experiencing toxicity caused by the use of GO particles in potential bioapplications.