Designing safer nanohybrids: stability and ecotoxicological assessment of graphene oxide-gold nanoparticle 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% 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.