In Vitro Antitumor Effects of Melittin Attached to Fe3O4 Magnetic Nanoparticles with Synergistic Contribution of Magnetic Hyperthermia

Melittin (Mel) is a membrane-active peptide with potential anticancer activity, but its direct therapeutic application may be limited by nonspecific toxicity and delivery-related challenges. The study aimed to assess melittin-functionalized magnetic nanoparticles (MNPs-Mel) as a strategy to enhance antitumor activity in Caco-2 cells, with/without magnetic hyperthermia (MH) association. BJ fibroblasts were used as a normal human in vitro cellular model. The effects of free Mel (2.5 µg/mL), MNPs, and MNPs-Mel (50 µg/mL both) + MH (30 min at 355 kHz and 25 kA/m) were assessed using colorimetry (for viability), luminescence (ATP), and spectrophotometry (lactate) following different exposure conditions. The mechanism of apoptosis induction was evaluated by ELISA (caspase 8 and 9 levels). Transmission electron microscopy (TEM) was also used to evaluate nanoparticle morphology and treatment-associated cellular ultrastructural changes. Free Mel reduced viability in both cell lines, with Caco-2 cells showing greater sensitivity at lower concentrations. MNPs (with/without MH) produced limited and less consistent effects, whereas MNPs-Mel significantly reduced Caco-2 viability and ATP levels and increased LDH and caspase 9. MH further enhanced the effects of MNPs-Mel: reduced viability (57–58% of the control at 24 h and 72 h), decreased ATP levels (67% of the control at 24 h and 53% at 72 h), increased LDH levels (206% of the control at 24 h and 301% at 72 h), and induced the mitochondrial apoptotic pathway (caspase 9 increased with 2164% of the control at 72 h). TEM proved the internalization of both MNPs and MNPs-Mel and revealed extensive ultrastructural alterations concerning mitochondria and lysosomes produced by MNPs-Mel, particularly in the Caco-2 cells. These modifications were heavily increased by MNPs-Mel + MH exposure. Overall, these findings demonstrate that Mel functionalization increases the antitumor activity of Mel at lower doses and that MH further potentiates this effect in Caco-2 cells.