A pH-sensitive nanococktail enabled cancer stem cell elimination, deep tumour penetration, and tumour shrinkage.

Cancer stem cells (CSCs) are intrinsically resistant to conventional therapies and play a pivotal role in cancer metastasis and recurrence. Eradication of CSCs requires combination strategies that simultaneously target multiple pathways at therapeutical concentrations throughout tumours. This study investigated the potential of a fusogenic pH-sensitive liposomal (pSL) nanococktail comprising doxorubicin and bufalin for targeting CSCs in clinically relevant HER2-positive breast cancer models, including cell lines (CD44high HCC1954 and CD44low BT474), homotypic and heterotypic spheroids, patient-derived organoids, and orthotopic HCC1954 mouse xenografts. At the optimal doxorubicin: bufalin ratio (10:1), the pSL-cocktail demonstrated pronounced synergistic efficacy and significantly outperformed all control formulations, including monodrug-loaded pSLs, non-pH-sensitive (DOXIL-like) liposomes, and free drugs. It significantly suppressed tumour proliferation and stemness, including CSC-associated migration, mammosphere formation, and self-renewal. In spheroids, the nanococktail rapidly penetrated to the spheroid core (≥100 µm within 1 h) and suppressed invasive dissemination. Similarly, it disrupted patient-derived organoids, achieving > 90% reduction in ATP levels and > 90% cell death. In vivo, the pSL-cocktail was well-tolerated and achieved significant tumour shrinkage, whereas DOXIL-like liposomes and free drugs showed minimal activity or severe adverse effects. Overall, this pSL-cocktail demonstrates strong potential for CSC-elimination and tumour clearance through synergistic drug combination, superior tumour penetration, and endosomal pH-triggered intracellular drug release.