Magnetic hyperthermia (MH) is the selective temperature increase in tumour tissues preloaded with magnetic nanoparticles (MNP) able to release heat in response to application of an alternating magnetic field (AMF) in the radiofrequency range. Despite much research, the underlying mechanism as to how this treatment causes cell death remains unclear. To test whether the biological effects mediated by MNP under an AMF were caused by heat dissipation originated from MNP, we performed in vitro MH experiments using (3-aminopropyl)-triethoxysilane-coated MNP (APS-MNP) with different core sizes (6, 8 and 14 nm). Suspended in water, these MNP produce increasing amounts of heat in the presence of an AMF (30 mT maximum, 250 kHz). After characterizing the MNP, we studied cell viability and particle uptake by murine pancreatic adenocarcinoma Pan02 cells. We measured cytotoxicity using a colorimetric PrestoBlue assay, quantified intracellular MNP amounts by ICP-OES (inductively coupled plasma atomic emission spectroscopy), and visualized the MNP by TEM (transmission electron microscopy). In addition, we used the PrestoBlue assay to test the effect of MH treatment on cell viability. To complement these results, we analysed activation of the effector caspase-3 by Western blot, and studied changes in cell morphology by TEM. After AMF exposure, cell viability was reduced. TEM images showed morphological features of apoptosis, which was confirmed by caspase-3 activation. We nonetheless found that all three MNP showed similar results, despite their distinct heat-producing abilities. Comparison of mRNA Hspa1b levels (increased in stress conditions, including heat stress) showed increased expression not only by MNP-loaded cells but also by non-loaded control samples when exposed to AMF. Cells thus received heat from an external source, the copper coil that generated the AMF and was heated due to passage of the electric current. To resolve this difficulty, experimental settings were modified and MH experiments repeated, maintaining the culture medium temperature at 37 ºC throughout AMF application, thus excluding the effect of the heat generated by the coil. In the conditions of these new experimental settings, we found none of the previously observed effects, in agreement with results described in the literature [1,2]. In conclusion, in our experimental setting MH treatment only appears to have an effect when combined with external heating. Our goal now is to understand our system when applying magnetic hyperthermia under physiological temperature conditions.

PHF received a pre-doctoral Severo Ochoa grant from the Department of Research, Development and Innovation, within the Spanish Ministry of Economy and Competitiveness (MINECO). MT received a JdC post-doctoral grant (JCI-2012-13159) from the MINECO. This work was supported by a grant from the MINECO (SAF-2014-54057-R to DFB).

Poster presented at the Nanomaterials Applied to Life Sciences (NALS), held in Gijón (Spain) on December 13-15th, 2017.