Designing safer nanohybrids: stability and ecotoxicological assessment of graphene oxide–gold nanoparticle hybrids in embryonic zebrafish

Abstract

Graphene oxide (GO) and graphene oxide–gold (GO–Au) nanohybrids offer promising applications in nanomedicine, biosensing, and environmental technology due to their unique properties. However, concerns regarding their environmental and biological safety remain largely unexplored. This study, using a safe and sustainable by design (SSbD) approach, evaluates the cytotoxicity, oxidative stress, and dispersion stability of GO and GO–Au nanohybrids in zebrafish ZF4 cells. GO was synthesised using a modified Hummer's method and GO–Au nanohybrids were prepared by incorporating gold nanoparticles (AuNPs) into the GO matrix. Physicochemical characterisation revealed enhanced dispersion stability of GO–Au nanohybrids, retaining over 98% of their initial absorbance in ultrapure water (UPW) and over 95% in DMEM/F12 after 48 hours. In contrast, GO displayed higher levels of sedimentation. Toxicity assessments indicated a dose- and time-dependent decrease in cell viability. After 72 hours, ZF4 cell viability was reduced to 39.5% for 150 μg mL⁻¹ GO, whereas GO–Au treatment at the same concentration exhibited a less severe reduction (54.5% viability). Reactive oxygen species (ROS) generation was significantly higher in GO-treated cells compared to GO–Au, with GO generating approximately 2x more ROS at concentrations of 50 μg mL⁻¹ and 100 μg mL⁻¹. Apoptosis and necrosis rates were also significantly elevated in GO-treated cells, with necrosis reaching 53.1% at 100 μg mL⁻¹, compared to 14.6% in GO–Au-treated cells. These findings demonstrate that the incorporation of AuNPs reduces cytotoxicity and oxidative stress by enhancing the colloidal stability of GO–Au nanohybrids. This study provides critical baseline data on the interaction of GO-based nanomaterials (NMs) with biological systems and highlights the importance of NM modification for safer, more sustainable applications.