Chitosan nanoparticles co-delivering curcumin and methotrexate synergistically induce p53/caspase-9-mediated apoptosis in metastatic breast cancer and effectively inhibit tumor growth in vitro and in vivo

• pH-sensitive chitosan NPs co-deliver curcumin/MTX with >80% encapsulation efficiency. • Cur-/MTX-Cs NPs induce 62.92% apoptosis via P53/caspase-9 in 4T1 breast cancer. • NPs reduce 4T1 cell migration by 56.7% and angiogenesis by 68.9% in vitro. • Cur-/MTX-Cs NPs achieve 91.12% tumor growth inhibition in vivo, outperforming free drugs. • Biocompatible NPs show minimal systemic toxicity in BALB/c mice. Nanotechnology offers innovative solutions to enhance cancer treatment efficacy through passively- or actively-targeted drug delivery systems. This study developed curcumin (Cur)- and methotrexate (MTX)-loaded chitosan (Cs) nanoparticles (NPs) using ionic gelation to treat 4T1 breast cancer cells, a highly metastatic model. The NPs, characterized by sizes <100 nm, zeta potentials <27 mV, and encapsulation efficiencies >80%, were biocompatible and significantly enhanced the cytotoxicity of Cur and MTX. Synthesized via a scalable, cost-effective ionic gelation method, these NPs leverage chitosan’s pH-sensitive properties to achieve synergistic drug release in acidic tumor microenvironments, offering a versatile platform for combination therapies. In vitro assays, including MTT, flow cytometry, scratch, mammosphere, and chorioallantoic membrane (CAM) tests, demonstrated that Cur-/MTX-Cs NPs induced 62.92% apoptosis (predominantly late-stage), reduced cell migration by 56.70% at 72 h, disrupted mammospheres by 20.36%, and inhibited angiogenesis by 68.9%. Western blotting revealed upregulated p53 expression and a 72.17% increase in caspase-9 activity, underscoring the apoptotic mechanism. In vivo, Cur-/MTX-Cs NPs achieved 91.12% tumor growth inhibition (TGI) in BALB/c mice bearing 4T1 tumors, significantly (p = 0.001) outperforming free Cur and MTX (38.07% TGI), with minimal systemic toxicity as confirmed by hematological, biochemical, and histopathological analyses. These findings highlight Cur-/MTX-Cs NPs as a safe and effective drug delivery system for breast cancer therapy, with potential for clinical translation to improve patient outcomes across diverse cancer types.