Enhanced therapeutic action of Trastuzumab loaded ZnxMn1−xFe2O4 nanoparticles using a pre-treatment step for hyperthermia treatment of HER2+ breast cancer

In this study, Ferrites (Fe<inf>3</inf>O<inf>4</inf>, MnFe<inf>2</inf>O<inf>4</inf>, ZnFe<inf>2</inf>O<inf>4</inf>) and different stoichiometric ratios of Zn<inf>x</inf>Mn<inf>1−x</inf>Fe<inf>2</inf>O<inf>4</inf> (x = 0.2, 0.4, 0.6, and 0.8) nanoparticles (<15 nm) were synthesized by microwave-assisted method and optimised for hyperthermia studies. The selection of the optimised variant of ferrite i.e. Zn<inf>0.4</inf>Mn<inf>0.6</inf>Fe<inf>2</inf>O<inf>4</inf> was found to be the best variant based on VSM (38.14 emu g⁻¹) hyperthermia-based temperature rise (maximum ΔT of 38 °C), SAR and ILP values. Trastuzumab, which is known to bind with HER2 receptors of breast cancer was chemically tethered onto Zn<inf>0.4</inf>Mn<inf>0.6</inf>Fe<inf>2</inf>O<inf>4</inf> nanoparticles through EDC/NHS coupling with a loading efficiency of 80%. The attached Trastuzumab aided during the pre-treatment step by aiding in the internalisation of Zn<inf>0.4</inf>Mn<inf>0.6</inf>Fe<inf>2</inf>O<inf>4</inf> nanoparticles, with cellular uptake of 11% in SK-BR-3 (cancerous HER2+) cells compared to ∼5% for MDA-MB-231 (cancerous HER2-) and RPE-1 (non-cancerous) cells. In the presence of a hyperthermia trigger for 15 mins, Zn<inf>x</inf>Mn<inf>1−x</inf>Fe<inf>2</inf>O<inf>4</inf> -Trastuzumab formulation had a maximum therapeutic effect by reducing the SK-BR-3 cell viability to 14% without adversely affecting the RPE-1 cells. The mechanism of Zn<inf>x</inf>Mn<inf>1−x</inf>Fe<inf>2</inf>O<inf>4</inf>-Trastuzumab combination was examined using an internalisation study, MTT-based viability, proliferation study, and ROS generation assay. By utilizing both Trastuzumab and hyperthermia, we achieve their synergistic anticancer properties while minimizing the drug requirement and reducing any effect on non-cancerous cells.