Designing Safer Nanohybrids: Stability and Ecotoxicological Assessment of Graphene Oxide-Gold Nanoparticles Hybrids in Embryonic Zebrafish

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% at 150 µg/mL of 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 and 100 µg/mL. Apoptosis and necrosis rates were also significantly elevated in GO-treated cells, with necrosis reaching 53.1% at 100 µg/mL, compared to 14.6% in GO-Au-treated cells. The findings demonstrate that the incorporation of AuNPs reduce 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 NMs modification for safer, more sustainable applications.