Smart thermo/pH‑responsive Janus nanogel for synergistic chemo-magnetic hyperthermia therapy inducing caspase‑mediated apoptosis in the BALB/c 4T1 breast cancer model.

Effective treatment of aggressive breast tumors remains challenging due to poor drug selectivity, systemic toxicity, and limited therapeutic synergy. Here, a thermo/pH‑responsive magnetic Janus nanogel was employed as a smart platform for on‑demand co‑delivery of 5‑fluorouracil (5‑Fu) and quercetin (Qu) combined with magnetic hyperthermia. Drug release behavior under acidic and hyperthermic conditions was investigated, followed by evaluation of cellular uptake and cytotoxicity in 4T1 breast cancer cells. Therapeutic efficacy was further assessed in BALB/c mice bearing orthotopic 4T1 tumors under alternating magnetic field (AMF)-induced hyperthermia. Tumor growth, histopathological alterations, hepatotoxicity, and the expression of apoptosis‑related genes (Bax, Bcl‑2, p53, caspase‑3, caspase‑8, and caspase‑9) were analyzed. The Janus nanogel exhibited dual-stimuli-responsive release of 5-Fu and Qu and showed enhanced in vitro cytotoxicity in the nanoformulated co-delivery system. In vivo, treatment with drug‑loaded nanogels combined with AMF produced pronounced tumor regression (p < 0.01) and extensive apoptotic cell death. Gene expression analysis indicated activation of both intrinsic and extrinsic apoptotic pathways, reflected by upregulation of Bax, p53, and caspases together with an increased Bax/Bcl‑2 ratio. No significant hepatotoxicity was observed. The integration of dual‑drug chemotherapy with magnetic hyperthermia using a thermo/pH‑responsive Janus nanogel provides a synergistic and safe therapeutic strategy for aggressive breast cancer. These findings highlight the potential of stimuli‑responsive nanocarriers for advanced combination cancer therapy.