Adverse Effects of Fullerenes on Endothelial Cells: Fullerenol C₆₀(OH)₂₄ Induced Tissue Factor and ICAM-1 Membrane Expression and Caused Apoptosis In Vitro.

Due to recent advances in nanotechnology, it is expected that carbon nanostructures will soon be used for different medical applications including devices for transfusion medicine. Fullerenes and carbon nanotubes have a profound impact on the development of diagnostic biosensors, drug delivery nanosystems, or imaging nanoprobes for intravascular use. In addition, fullerenes can also be used as components of plastic and filtration membranes. Furthermore, fullerene C₆₀ has been shown to have antiviral and antibacterial properties and its photodynamic potential for pathogen-reduction treatment of blood products has been suggested. Hydroxylated C₆₀ derivatives have potent antioxidant properties, however, there are concerns about possible cytotoxic effects of fullerenes and/or their oxidative products. We studied the effects of C₆₀ on human umbilical vein endothelial cells (HUVECs) in culture. We used a water suspension of nonsoluble C₆₀ (C₆₀ preparations of 99.5% and 99.9% purity from SES Res., Houston, TX, and MER Corp., Tuscon, AZ, respectively) at 4 μg/mL, particle size < 500 nm, and the water soluble polyhydroxylated fullerene derivative fullerenol C₆₀(OH)₂₄ at 1 – 100 μg/mL (MER Corp., Tuscon, AZ). We found that 24 hr treatment of HUVECs with C₆₀(OH)₂₄ at 100 μg/mL significantly increased cell surface expression of ICAM-1(CD54) (67±4%CD54⁺ cells vs. 19±2 % CD54⁺ cells in control; p< 0.001). Moreover, this treatment induced expression of tissue factor (CD142, detected by HTF-1 Mab) on HUVECs (54±20% CD142⁺ cells vs. 4±2% CD142⁺ cells in control; p=0.008) and increased exposure of phosphatidylserine (PS, detected by Annexin V) (29±2% PS⁺ cells vs. 12±5% PS⁺ cells in control; p<0.001). In addition, using the MTS proliferation assay, we found that C₆₀(OH)₂₄ significantly inhibited HUVEC proliferation (35± 8% inhibition at 10 μg/mL). Analysis of cell cycle and DNA fragmentation (TUNEL) by flow cytometry showed that both C₆₀ and C₆₀(OH)₂₄ caused G1 arrest of HUVECs and C₆₀(OH)₂₄ induced significant apoptosis (21±2% TUNEL⁺ cells at 100 μg/mL of C₆₀(OH)₂₄ vs. 4±2% TUNEL⁺ cells in control; p<0.001). The acute effect of fullerenes on intracellular free Ca²⁺ concentration [Ca²⁺]_i was studied, using a ratio fluorometry in HUVECs, GT1-7 and PC12 cells. Cells were loaded with a Ca²⁺-sensitive probe FURA-2AM. We demonstrated that both C₆₀ and C₆₀(OH)₂₄ induced a rapid concentration dependent elevation of [Ca²⁺]_i. For example, C₆₀(OH)₂₄ at 100 μg/mL caused 149±30 nM increase in [Ca²⁺]_i in HUVECs. The activity could be inhibited by EGTA, suggesting that the source of [Ca²⁺]_i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In contrast, fullerenes tested at the given concentrations, did neither induce platelet aggregation nor affect TRAP or ADP/epinephrine-induced platelet aggregation, as tested with human PRP. In conclusion, our results indicate possible adverse effects of fullerenes on the endothelium. Hydroxyfullerene C₆₀(OH)₂₄, which may be formed as an oxidative product of C₆₀ fullerenes, inhibited cell proliferation and had both proinflammatory and proapoptotic effects on endothelial cells. These findings warrant further studies on vascular biocompatibility of carbon nanostructures. The views of the authors represent their scientific opinion and should not be construed as FDA policy.