Three Noble Metal Nanoparticles Demonstrated Distinct Biological Interactions and Cell Death Pathways in Human Hepatocarcinoma Cell Lines.

Purpose: New, more effective therapies are needed for patients with liver tumors who are ineligible for surgical resection, transplantation, or local ablation. Noble metal nanoparticles (NPs) exhibit substantial biological reactivity toward liver cancer cells, yet their unpredictable nanotoxicity highlights the need for a deeper understanding of NP-cell interactions. The present study examined the in vitro toxicological properties of gold (Au), palladium (Pd), and Pd-Au NPs in three hepatocellular carcinoma cell lines (HepG2, Hep3B, and Huh7D-12). Methods: The cytotoxicity of noble-metal-based NPs was assessed using Alamar Blue and MTT colorimetric assays. Reactive oxygen species generation and oxidative stress markers were quantified by fluorometric, luminometric, and flow cytometry methods. Confocal microscopy combined with fluorescence readouts were used to evaluate mitochondrial homeostasis and programmed cell death pathways. Molecular markers of NP-induced cellular stress were further confirmed using qRT-PCR. Results: Distinct cellular differences were observed among the three NPs, particularly in terms of cytotoxicity and death modalities. Au NPs caused mild mitochondrial perturbations without significant cytotoxicity. Pd NPs induced necroptosis, primarily at high concentrations. In contrast, Pd-Au NPs induced oxidative stress, lipid peroxidation, glutathione depletion, and transcriptional changes in the genes regulating glutathione synthesis and metabolism. Pd-Au NPs also activated ferroptosis, which is a regulated cell death pathway increasingly recognized as a promising anticancer strategy. Conclusion: Among the tested formulations, Pd-Au NPs exhibited the strongest cytotoxic and antiproliferative effects that were driven by mitochondrial disruption and ferroptosis induction. These in vitro findings provided a mechanistic reason for advancing Pd-Au NP research efforts into in vivo studies and patient-derived preclinical models for hepatocellular carcinoma.