Background: Combination therapies for cancer have gained considerable attention due to their potential for enhancing therapeutic efficacy and decreasing drug resistance. Introducing nanodrug delivery systems in this context may further improve the therapy due to targeted delivery, improved drug stability, sustained drug release, and prevention of rapid clearance from circulation. This study evaluates the combinatorial effects of two cytotoxic drugs, cabazitaxel (CBZ) and RSL3 (RAS-selective lethal 3), in free form as well as encapsulated within poly(2-ethyl butyl cyanoacrylate) (PEBCA) nanoparticles (NPs) in breast cancer cell lines. Methods: Cell proliferation was assessed using IncuCyte technology, and synergistic drug effects were determined with SynergyFinder Plus. Cell viability was measured with the MTT assay. Additionally, we investigated whether the combinatorial effects were reflected in alterations of metabolic activity or reactive oxygen species (ROS) production using Seahorse technology and the CM-H₂DCFDA assay, respectively. Results: The data presented reveal, for the first time, that CBZ and RSL3 exhibit synergistically or additively combinatorial effects on various breast cancer cell lines. The pattern of cytotoxic effects was consistent, whether the drugs were in free form or encapsulated in NPs. Moreover, the combinatorial effects were not observed to be associated with early changes in metabolic activity or ROS production. Conclusion: This study highlights the potential of CBZ and RSL3 in combinatorial nanomedicine as they may act synergistically. Further studies are warranted to better understand the mechanisms behind these combinatorial effects.

Breast cancer positive for human epidermal growth factor receptor‐2 (HER2) is challenging to treat due to the development of drug resistance, even with recently developed antibody‐mediated therapeutics. However, nanotechnology provides new innovative therapeutic strategies. This study evaluates the efficacy of new HER2‐targeted, pH‐sensitive poly(ethylene glycol)‐poly(L‐histidine) copolymer micelles (PEG‐PHis micelles), either empty, loaded with the drug doxorubicin (DOX), or the fluorophore Rhodamine‐B (RB) for imaging purposes. Cellular association of the micelles, in vitro effects on proliferation and cytotoxicity, as well as ex vivo and in vivo responses on tumor growth are assessed. The results indicate that trastuzumab (TZM) conjugation enhances the specific cellular association of PEG‐PHis micelles in HER2 overexpressing cells and improves the cytotoxic effects of the micelles themselves. Although the TZM conjugation of DOX‐loaded micelles did not contribute to an enhancement in overall efficacy in cell lines and tumor explant cultures, the findings highlight the potential of TZM conjugation on these micelles in targeting HER2‐positive tumors.