Combination chemotherapy is often limited by the distinct physicochemical properties and intracellular behaviors of individual drugs. This limitation leads to poorly coordinated delivery in tumor tissues. In this study, pH-responsive ionizable nanoliposomes were developed using the ionizable lipid DLin-DMA to co-deliver erlotinib and doxorubicin hydrochloride. This approach introduces a novel spatial and temporal synchronization strategy for two mechanistically distinct drugs within a single carrier. The resulting nanoliposomes formed stable nanoscale assemblies with an optimized particle size of 139.8 nm and high encapsulation efficiencies of 84.8% for erlotinib and 80.8% for doxorubicin hydrochloride. The formulation exhibited a pronounced pH-dependent surface charge transition. It remained near neutral under physiological conditions but converted to a cationic state in acidic environments relevant to tumors. This charge conversion triggered a sequential drug release profile, featuring an immediate accelerated release of doxorubicin hydrochloride for rapid cytotoxicity alongside a steady sustained release of erlotinib for prolonged targeted inhibition. In MDA-MB-231 triple-negative breast cancer cells, this coordinated cellular internalization and subcellular distribution resulted in profound synergistic cytotoxicity, which was quantitatively confirmed by a Combination Index of 0.427. In vivo evaluations using an MDA-MB-231 xenograft model further demonstrated that the charge-conversional mechanism significantly enhanced tumor accumulation and deep tissue penetration following systemic administration. This targeted accumulation led to robust tumor growth suppression without pronounced systemic toxicity. Overall, this study demonstrates how ionizable lipid chemistry can orchestrate the intracellular fate of chemically dissimilar small molecule drugs, providing a highly effective materials-based framework for synergistic combination therapy in solid tumors.
