Multi-Targeting Effects and Orchestrated Induction of Multiple Cell Death Modalities by Silver–Copper Alloy Nanoparticles Functionalized Drug Nanocrystals in Cancer Cells

To combat multidrug resistance and cancer stem cell (CSC) persistence, we constructed a tumor-targeted nanoplatform integrating silver/copper alloy nanoparticles (Cu-Ag NPs) and camptothecin (CPT) nanocrystals for synergistic multimodal therapy. The nanocomposite was fabricated by stepwise assembly of CPT nanocrystals, a polydopamine coating, and functionalization with Cu-Ag NPs plus a tumor-mitochondria dual-targeting peptide. It exhibited a hydrodynamic diameter of ∼152.67 nm, high colloidal stability, favorable photothermal performance, and pH/NIR-responsive drug release. Under NIR irradiation, it showed potent and selective cytotoxicity against triple-negative breast cancer cells (IC₅₀ = 16.92 ± 0.22 μg/mL), with strong synergy (CI < 0.3) between inorganic Cu-Ag NPs and organic CPT. Actively targeting both cancer cells and mitochondria, it induced severe mitochondrial dysfunction─loss of MMP, ATP depletion, ROS burst, and mtDNA damage. Moreover, it acted as a potent cuproptosis inducer via exogenous copper, evidenced by FDX1 and DLAT downregulation (48.23% and 68.61%) and HSP70 upregulation (61.42%). Additional cell death pathways, including apoptosis, necrosis and pyroptosis, were also activated through nuclear DNA damage and plasma membrane rupture. Importantly, this nanoplatform effectively targeted stubborn breast CSCs, exhibiting an IC₅₀ as low as 13.70 ± 0.36 μg/mL─attributed to the mitochondrial targeting and subsequent inhibition of robust oxidative phosphorylation within CSCs, which rely more heavily on this pathway than on glycolysis compared to conventional cancer cells. In summary, this work presents a novel "multi-targeting" therapeutic strategy that orchestrates mitochondrial dysfunction, cuproptosis, apoptosis, and pyroptosis via a chemo-photothermal combination, offering a robust and broad-spectrum approach to eradicate both conventional resistant cancer cells and refractory CSCs.