release from arthritis-related cells including MC and synovial fibroblasts, as well as their effect on human osteoclast formation. The best candidates were selected for their ability to attenuate inflammatory arthritis in vivo using the K/BxN serum transfer arthritis and CIA [29,30]. It is demonstrated that the ability of fullerene derivatives to inhibit inflammatory cell mediator release was dependent on the moieties added to the carbon cage. The ability to inhibit in vitro mediator release translated to in vivo efficacy only in the K/ BxN induced mice, but this effect was independent of the presence of MC. CIA mice showed no reduction in disease onset or progression when treated with fullerene derivatives. Of particular interest, dye-conjugated fullerene derivatives localized specifically to inflamed joints, and did not accumulate in other organs. Our results suggest rationally designed fullerene derivatives may provide an effective therapeutic option for the treatment of inflammatory arthritis by targeting ROS to prevent stimulation of pro-inflammatory cytokines, osteoclast formation, and stabilizing critical cells involved in RA progression.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the International Animal Care and Use Committee of the University of North Carolina at Greensboro (Protocol Number: 112). All techniques were performed under isoflurane anesthesia, and all efforts were made to 
minimize suffering. A panel of fullerene derivatives was synthesized at Luna Innovations and characterized for particle size using dynamic light scattering (Malvern Instruments, Zetasizer Nano ZS, Westborough, Massachusetts, USA), qNano (Izon Science, qNano, Cambridge, Massachusetts, USA) and nano particle tracking analysis, (Malvern Instruments, Nanosight LM10, Westborough, Massachusetts, USA), zeta potential (Malvern Instruments, Zetasizer Nano ZSP, Westborough, Massachusetts, USA), NMR (Aglafolin Agilent Technologies, 400 Mhz NMR Spectrometer, Santa Clara, California, USA), and FT-IR (Agilent Technologies, Varian 670 FT-IR, Santa Clara, California, USA). A representative physiochemical characterization schematic for the two fullerene derivatives used for the in vivo studies (ALM, a liposome encapsulated C70 fullerene and TGA, a water-soluble C70 fullerene conjugated with four glycolic acids) is shown in [27,31].
The activation of MC through Fc receptors is one constituent that drives inflammatory arthritis. Unlike primary human MC, mouse MC express FcRIII and may be stimulated to release mediators via this receptor in culture, although activation is constrained by the inhibitory receptor FcRII [32]. Thus, BMMC were generated from FcRII-deficient mice by incubation for at least 4 weeks in SCF (12.5 ng/ml) and IL-3 (10 ng/ml)-containing medium [29]. Cells were incubated with or without fullerene derivatives overnight [10 g/ml; [25]], washed and rat anti-FcRII/III (2.4G2, 1g/ml) added for two hours. Immune complex (IC) stimulation was mimicked by the addition of donkey anti-rat (DAR; 1 g/ml) for 30 minutes (degranulation) or 24 hours (cytokine) and cellular lysates prepared [33] collected and IL-1 measured using ELISA as described [25]. Human connective tissue MCTC [34] were generated from skin and stimulated with immune complexes (IC) and mediator release measured as described [35]. Initial experiments deter