Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.