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.

The present study describes for the first time a procedure for the radiolabeling of fullerene (C(60)) nanocrystals (nanoC(60)) with Na (125)I, as well as the biodistribution of radiolabeled nanoC(60) ((125)I-nanoC(60)). The solvent exchange method with tetrahydrofuran was used to make colloidal water suspensions of radiolabeled nanoC(60) particles. The radiolabeling procedure with the addition of Na (125)I to tetrahydrofuran during dissolution of C(60) gave a higher radiochemical yield of radiolabeled nanoC(60) particles in comparison to the second option, in which Na (125)I was added after C(60) was dissolved. Using photon correlation spectroscopy and transmission electron microscopy, (125)I-nanoC(60) particles were found to have a crystalline structure and a mean diameter of 200-250 nm. The (125)I-nanoC(60) had a particularly high affinity for human serum albumin, displaying 95% binding efficiency after 1 h. Biodistribution studies of (125)I-nanoC(60) in rats indicated significant differences in tissue accumulation of (125)I-nanoC(60) and the radioactive tracer Na (125)I. The higher accumulation of radiolabeled nanoC(60) was observed in liver and spleen, while accumulation in thyroid, stomach, lungs and intestines was significantly lower in comparison to Na (125)I. In addition to being useful for testing the biological distribution of nanoC(60), the described radiolabeling procedure might have possible applications in cancer radiotherapy.