Antitumor Effects of Melittin-Functionalized Fe3O4 Magnetic Nanoparticles in Collagen-I-Based Caco-2/BJ/HUVEC Spheroids Enhanced by Magnetic Hyperthermia

This study evaluated the integrated biochemical and ultrastructural response of three-dimensional colorectal cancer spheroids to magnetic hyperthermia (MH) mediated by melittin-functionalized Fe3O4 magnetic nanoparticles (MNPs-Mel). The spheroids comprised human Caco-2 colorectal adenocarcinoma cells, dermal fibroblasts (BJ), and human umbilical vein endothelial cells (HUVECs) in a ratio of 3:1:1 in a collagen-I matrix. They were assigned to control, alternating magnetic field (AMF) control (Control-MH), free melittin (Mel), unloaded MNPs, MNPs-MH, MNPs-Mel, and MNPs-Mel-MH groups and assessed at 24 and 72 h. MTS-reducing activity (colorimetry) and ATP-associated luminescence were quantified as viability markers, while VEGF, HIF-1α, and caspase-9 concentrations measured by ELISA were used to assess angiogenesis and apoptosis induction. MNPs’ size, shape and internalization and treatment-associated ultrastructural changes were examined by transmission electron microscopy (TEM). Treatment, time, and their interaction significantly affected all biochemical endpoints. Every intervention reduced MTS absorbance relative to control at both time points. MNPs-Mel produced lower MTS values than unloaded MNPs, whereas MNPs-Mel-MH generated the lowest ATP signal at both time points and the lowest MTS signal at 72 h. The combined treatment also reduced VEGF and increased caspase-9, maximal at 72 h, while the increase in HIF-1α at 24 h was not maintained at 72 h. TEM showed intracellular MNPs-Mel and a more restricted distribution within spheroids than in the separately examined monocultures. Free Mel and unloaded MNPs produced time-dependent ultrastructural changes, while MH accentuated the changes associated with MNP exposure. MNPs-Mel-MH led to pronounced mitochondrial polymorphism, cristae loss, matrix rarefaction, autophagic-lysosomal alterations, endoplasmic reticulum expansion, reduced intercellular adherence, and extensive disruption of Caco-2 cells, particularly at 72 h. However, biochemical endpoints represent the response of the whole spheroid and do not distinguish effects on Caco-2 cells from those on BJ fibroblasts or HUVECs. The molecular and ultrastructural findings identify MNPs-Mel-MH as the treatment associated with the broadest response in this tri-cellular spheroid model, linking sustained metabolic suppression and reduced pro-angiogenic signaling with marked mitochondrial and cellular injury.