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

Abstract

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.

Figure 1

Representative photographs for vial containing the as-prepared dispersions for graphite oxide before exfoliation (a), graphite oxide after mechanical exfoliation (b), GO prepared from the exfoliation of graphite oxide and suspended in water with 5 min of ultrasonication (c), and GO suspended in DMEM (supplemented with 10% FBS) (d).

Figure 2

UV–vis absorbance spectra for GO (a) and RGO (b) solutions in water. The inset plots show the absorbance per unit path length (A/l) for several concentrations (in mg/mL) measured at the maximum peak intensity following the Lambert–Beer law. The extinction coefficients for GO and RGO are obtained from linear regressions. Dynamic light scattering measurements in water for GO (c) and RGO flakes (d) after exfoliation, lyophilization, and ultrasonication. Zeta-potential of particles (in mV) are shown in the inset plots.

Figure 3

XPS for GO (left) and RGO (right) particles showing the characteristic peaks associated with for O 1s, N 1s, and C 1s transitions (a). The atomic percentage for the content of oxygen is calculated in about 21.5% and 13.3% for GO and RGO, respectively. The synthetic components related to different oxygen functionalities are shown for GO (b) and RGO (c) for both O 1s and C 1s transitions. Residual STD is the value obtained after the deconvolution process for obtaining the components. The XPS spectra were obtained with erosion using Ar to detach the excess of physisorbed O₂.

Figure 4

Hypertrophic effect of angiotensin on H9c2 myoblasts. (a) Representative images from confocal microscopy of H9c2 control cells (Ctrl, left) and H9c2 cells stimulated with 1 μM angiotensin II for 48 h (Ang II, right). Cytoplasm is shown in green (calcein) and nuclei in blue (Draq5). Scale bar represents 50 μm. (b) Pooled data from cell surface area. Gene expression of remodeling and inflammatory markers: BNP [Nppb gene, (c)], collagen type 1a [Col1a1 gene, (d)], and IL-6 [Il6 gene, (e)]. (f) Mitochondrial ROS production analyzed by flow cytometry. Data are presented as mean ± SEM; one-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Dose-dependent metabolic activity of H9c2 healthy cells (a) and H9c2 cells treated with Ang II (b) after 24 h exposure to different GO and RGO dispersion in DMEM. Viability percentages normalized to the control mean. The experiment presented as the mean ± SE, for n = 5 independent experiments.