Despite the rapid expansion of the biomedical applications of graphene oxide (GO), safety issues related to GO, particularly with regard to its effects on vascular endothelial cells (ECs), have been poorly evaluated. To explore possible GO-mediated vasculature cytotoxicity and determ
ine lateral GO size relevance, we constructed four types of GO: micrometer-sized GO (MGO; 1089.9 ± 135.3 nm), submicrometer-sized GO (SGO; 390.2 ± 51.4 nm), nanometer-sized GO (NGO; 65.5 ± 16.3 nm), and graphene quantum dots (GQDs). All types but GQD showed a significant decrease
in cellular viability
in a dose-dependent manner. Notably, SGO or NGO, but not MGO, potently
induced apoptosis while caus
ing no detectable necrosis. Subsequently, SGO or NGO markedly
induced autophagy through a process dependent on the c-Jun N-term
inal k
inase (JNK)-mediated phosphorylation of B-cell lymphoma 2 (Bcl-2), lead
ing to the dissociation of Becl
in-1 from the Becl
in-1–Bcl-2 complex. Autophagy suppression attenuated the SGO- or NGO-
induced apoptotic cell death of ECs, suggest
ing that SGO- or NGO-
induced cytotoxicity is associated with autophagy. Moreover, SGO or NGO significantly
induced
increased
intracellular calcium ion (Ca
2+) levels. Intracellular Ca
2+ chelation with BAPTA-AM significantly attenuated microtubule-associated prote
in 1A/1B-light cha
in 3-II accumulation and JNK phosphorylation, result
ing
in reduced autophagy. Furthermore, we found that SGO or NGO
induced Ca
2+ release from the endoplasmic reticulum through the PLC β3/IP
3/IP
3R signal
ing axis. These results elucidate the mechanism underly
ing the size-dependent cytotoxicity of GOs
in the vasculature and may facilitate the development of a safer biomedical application of GOs.
Statement of Significance
Graphene oxide (GO) have received considerable attention with respect to their utilization in biomedical applications. However, GO-related safety issues concerning human vasculature are very limited. In this manuscript, we report for the first time the differential size-related biological effects of GOs on endothelial cells (ECs). Notably, Subnanometer- and nanometersized GOs induce apoptotic death in ECs via autophagy activation. We propose a molecular mechanism for the GO-induced autophagic cell death through the PLCβ3/IP3/Ca2+/JNK signaling axis. Our findings could be provide a better understanding of the GO sizedependent cytotoxicity in vasculature and facilitate the future development of safer biomedical applications of GOs.