Equal nano-characteristics, unequal harm: the chemical composition of noble metal nanoparticles as the main factor of cytotoxicity

Background Metal nanoparticles are increasingly explored in biomedical and technological applications, yet their cytotoxicity remains difficult to interpret due to the strong influence of multiple physicochemical parameters. Differences in synthesis protocols, particle size, morphology, and surface properties across studies often hinder direct comparison of toxicological outcomes and limit the ability to attribute observed effects to the chemical composition of the nanoparticle core. Methods In this study, silver (Ag), copper (Cu), and gold (Au) NPs were synthesized under identical conditions to control for size, shape, and surface charge, thereby isolating the effect of chemical composition. The NPs (8–9 nm, spherical, zeta potential ∼ –21 mV) were evaluated in NIH 3T3 fibroblasts using MTT viability assays, reactive oxygen species (ROS) detection, mitochondrial membrane potential analysis, apoptosis quantification, and comet assays. Results Ag NPs showed the highest toxicity (IC 50 = 11.9 mg·L -1 ), followed by Cu NPs (51.6 mg·L -1 ), while Au NPs were the least toxic (228.2 mg·L -1 ). Mechanistic data revealed that Ag NPs induced severe oxidative stress and mitochondrial dysfunction, leading to apoptosis at sublethal concentrations and necrosis at higher doses. Cu NPs triggered strong ROS generation and apoptotic signaling. Au NPs showed minimal toxicity, with weak apoptotic effects only at the highest concentrations. None of the tested NPs caused significant DNA damage. Notably, cytotoxicity correlated with increased necrosis at higher doses and apoptosis at lower concentrations, indicating dose- and composition-dependent cell death mechanisms. While reactive oxygen species contributed to toxicity, the small size (∼8–10 nm), spherical morphology, and absence of surface modification enhanced cellular uptake and cytotoxic potential. Conclusion Our results provide clear evidence that, for controlled nanoparticle properties, the toxicity hierarchy primarily reflects intrinsic chemical identity. These findings underscore the importance of separating nanoparticle composition from other particle-related artifacts, thereby supporting the rational design of safer biomedical nanomaterials through the controlled adjustment of size, surface chemistry, and metal composition.