Novel Treatment of Radiculopathy with Fullerol Nanoparticles

Abstract

Introduction Back pain is ubiquitous and costs the United States an estimated $100 billion each year, second only to the common cold. Degeneration of intervertebral discs (IVDs) is considered to be the major cause of back pain, yet despite decades of efforts focused around treatment of disc degeneration, the predominant treatment option continues to be symptomatic care. Moreover, the mechanisms underlying degeneration are poorly lucidated. Compelling evidence suggests that reactive oxygen species (ROS) plays a pivotal role in disc degeneration. In disc tissues, oxidative stress may initiate or participate in matrix destruction and cell apoptosis that ultimately result in disc degeneration. Fullerol nanoparticles have been demonstrated outstanding ability to scavenge ROS. The goal of this study is to investigate the role of its antioxidative and anti-inflammatory in preventing and antagonizing disc degeneration. Materials and Methods Fullerol, a water soluble fullerene derivative, was characterized with UV-Vis spectrometry, dynamic light scattering, and zeta-potential analyses. The scavenging efficiencies against superoxide anions, hydroxyl radicals, and nitric oxide were determined by the pyrogallol auto-oxidation, Fenton-type reaction, and Griess reaction, respectively. For in vitro cellular experiments, a pro-oxidant H2O2 or an inflammatory cytokine interleukin (IL)-1β was employed to induce degenerated phenotypes in human nucleus pulposus (NP) cells encapsulated in alginate beads, and the effects of fullerol on matrix, ROS, and cell viability were assessed with real-time reverse-transcriptase polymerase chain reaction (RT-PCR), biochemistry, and immunofluorescence microscopy. In the animal study, an annulus degeneration model was induced with needle puncture method, and then 25 μL of fullerol (40 μg/mL) or phosphate-buffered saline (PBS) was injected intradiscally. The outcome was evaluated with magnetic resonance imaging (MRI), histology, and biochemistry. Results The absorption peak of fullerol suspension is 293 nm (UV-Vis spectrum), particle size is between 25 and 45 nm (dynamic light scattering), and average potential between − 40 and − 60 mV (zeta-potential analyzer). The nanofullerol suspension eliminated the radical and hydroxyl radicals in a dose-dependent manner, which is comparable to SOD and mannitol in high concentrations. Furthermore, nitric oxide was decreased by nanofullerol in a dose-dependent fashion. In alginate bead culture, H2O2 treatment significantly reduced the glycosaminoglycan content and the cellular amount of aggrecan in human NP cells. However, cotreatment with nanofullerol reversed the adverse effects of H2O2. Similar results were obtained in the case of type II collagen. While H2O2 caused a significant elevation in messenger RNA (mRNA) expression of matrix degradation enzymes MMP3, MMP9, and ADAMTS5, nanofullerol stopped the effect. The protective effects of fullerol on IL-1β-induced degeneration were also assessed in human NP cells. Human NP cells were treated with 10 ng/mL of IL-1β for up to 7 days in the presence or absence of fullerol. IL-1β caused an increase in nitric oxide secretion at day 3, as well as a decrease in cellular proteoglycan content at both day 3 and day 7. Fullerol elicited a significant counteraction against the IL-1b. Gene expression analysis by real-time RT-PCR showed that IL-1β induced a robust decrease in mRNA levels of aggrecan, type I and II collagens at day 7. Cotreatment with fullerol rescued the decreased mRNA expression of aggrecan and type II collagen but not type I. In addition, with nanofullerol itself, the expression of type II collagen was significantly upregulated. In the in vivo study, the anterior surfaces of rabbit lumbar IVDs were exposed under general anesthesia. Selected discs were punctured with an 18G needle and 25 µL of a nanofullerene suspension, or PBS, injected into the disc. The images from a morphological study and the X-ray and MRI scans showed that discs that were punctured and then injected with PBS showed a blurred annulus fibrosus (AF)/NP junction, narrowed disc space and a lower T2-weighted MRI signal, which indicated disc degeneration. However, discs punctured and then treated with fullerene showed a distinct AF/NP junction, improved disc height and strong T2-weighted MRI signals. Conclusion This study presented both in vitro and in vivo evidences supporting that the potent antioxidant fullerol rescues disc degeneration, which could be a novel therapeutic remedy for disc degeneration. Disclosure of Interest None declared