Nanoparticle induced hyperthermia has been considered as a promising approach for cancer treatment for decades. The local heating ability and drug delivery potential highlight a diversified possibility in clinical application, therefore a variety of nanoparticles that has been developed accordingly. However, currently, only a few of them have been translated into clinical stage indicating the ‘nanoparticle medically underserved’ situation, which encourages their comprehensive biomedical exploration. This study presents a thorough biological evaluation of previous well-developed dual pH- and thermo- responsive magnetic Doxorubicin-nanocarrier (MNC-DOX) in multiple cancer cell lines. The biocompatible of the nanocomposites has been proved by the MTT assay on primary cell lines. The histology and fluorescence microscopy imaging revealed the efficient but various cellular uptaking of nanocarriers among cell lines. The IC50 of MNC-DOX is significantly lower than free DOX without alternative magnetic field (AMF), which implied the potential to lower the systemic cytotoxicity in clinical research. The concurrent thermo-chemotherapy generated by this platform has been successfully achieved under alternating magnetic field (AMF). Promising effective synergistic results have been demonstrated through in vitro study in multi-model cancer cell lines via both trypan blue exclusion method and bioluminescence imaging method. Furthermore, the two most used magnetic hyperthermia modality, namely intracellular and extracellular treatments have been compared on the same nanocarriers in all 3 cell lines, which showed treatment after internalization is not required but preferable. These results lead to the conclusion that this dual responsive nanocarrier has extraordinary potential to serve as a novel broad-spectrum anticancer drug and worth to be pursued for potential clinical applications.Read less <