The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C₆₀) and water-soluble polyhydroxylated fullerene [C₆₀(OH)_n] were investigated. Crystal violet assay for cell viability demonstrated that nano-C₆₀ was at least three orders of magnitude more toxic than C₆₀(OH)_n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V–fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C₆₀ caused rapid (observable after few hours), reactive oxygen species (ROS)–associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C₆₀(OH)_n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C₆₀ toxicity, but not C₆₀(OH)_n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C₆₀(OH)_n-induced apoptosis, but not nano-C₆₀–mediated necrosis. Finally, C₆₀(OH)_n antagonized, while nano-C₆₀ synergized with, the cytotoxic action of oxidative stress–inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C₆₀ that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C₆₀ seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.

Magnetic nanoparticle (MNP)-mediated hyperthermia (MH) coupled with radiation therapy (RT) is a novel approach that has the potential to overcome various practical difficulties encountered in cancer treatment. In this work, we present recommendations for the in vitro and in vivo testing and application of the two treatment techniques. These recommendations were developed by the members of Working Group 3 of COST Action TD 1402: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy (“Radiomag”). The purpose of the recommendations is not to provide definitive answers and directions but, rather, to outline those tests and considerations that a researcher must address in order to perform in vitro and in vivo studies. The recommendations are divided into 5 parts: (a) in vitro evaluation of MNPs; (b) in vitro evaluation of MNP-cell interactions; (c) in vivo evaluation of the MNPs; (d) MH combined with RT; and (e) pharmacokinetic studies of MNPs. Synthesis and characterization of the MNPs, as well as RT protocols, are beyond the scope of this work.