This research investigates pH changes during the green synthesis of ZnO nanoparticles (NPs) and emphasises its importance in their physicochemical, antibacterial, and biological properties. Varying the synthesis pH from 8 to 12 using "Bravo de Esmolfe" apple extracts neither affected the morphology nor crystallinity of ZnO but impacted NP phytochemical loads. This difference is because alkaline hydrolysis of phytochemicals occurred with increasing pH, resulting in BE-ZnO with distinct phytocargos. To determine the toxicity of BE-ZnO NPs, Galleria mellonella was used as an alternative to non-rodent models. These assays showed no adverse effects on larvae up to a concentration of 200 mg/kg and that NPs excess was relieved by faeces and silk fibres. This was evaluated by utilising fluorescence-lifetime imaging microscopy (FLIM) to track NPs' intrinsic fluorescence. The antibacterial efficacy against Staphylococcus aureus was higher for BE-ZnO12 than for BE-ZnO8; however, a different trend was attained in an in vivo infection model. This result may be related to NPs' residence in larvae haemocytes, modulated by their phytocargos. This research demonstrates, for the first time, the potential of green synthesis to modulate the biosafety and antibacterial activity of NPs in an advanced G. mellonella infection model. These findings support future strategies to overcome antimicrobial resistance by utilizing distinct phytocargos to modulate NPs' action over time.
Keywords: FLIM; Galleria mellonella; Staphylococcus aureus; ZnO; apple; phytocargo.