Reductant-Responsive Squalene Dual-Prodrug Nanoparticles Co-Delivering Doxorubicin and Exatecan for Synergistic Breast Tumor Therapy

Background: combination chemotherapy employing agents with complementary mechanisms of action can enhance therapeutic efficacy and overcome resistance, but mismatched pharmacokinetics and systemic toxicity limit benefit. Methods: We designed reductant-responsive squalene prodrugs of doxorubicin (DOX) and exatecan (EXA) that co-assemble with DSPE-PEG2k into approximately 76 nm dual-prodrug nanoparticles (doxorubicin prodrug–exatecan prodrug nanoparticles, DP-EP NPs) at a predefined synergistic ratio 1:2. Results: The minimalist, drug-as-carrier formulation achieved high effective loadings with minimal leakage under normoxia, yet synchronously liberated both drugs in reductive, hypoxia-mimicking media (24 h release: DOX ~65% and EXA ~58% at 1 mM Na2S2O4 vs. ~8–11% at 0 mM Na2S2O4). In MCF-7 and 4T1 cells, the nanoparticles enhanced intracellular accumulation, intensified the disruption of DNA damage repair, as evidenced by increased colocalization of 53BP1 and enhanced γ-H2AX foci, and induced markers of immunogenic cell death, including increased CRT-associated cellular fluorescence and reduced intracellular HMGB1 staining. Furthermore, they partially reversed drug resistance in MCF-7/ADR cells. In rats, the formulation prolonged circulation and increased exposure (DOX t½ ~9×, AUC ~1.8×; EXA t½ ~5×, AUC ~14.9×) relative to the free-drug mixture. In 4T1 tumor-bearing mice, the nanoparticles produced the greatest tumor-growth inhibition with improved tolerability, supported by stable body weight and benign histopathology. Conclusions: These findings establish a scalable, excipient-lean platform that aligns pharmacokinetics with microenvironment-triggered pharmacodynamics to deliver synchronized Topo II/I inhibition for synergistic chemotherapy.