Manganese dioxide nanoparticles modulate oxidative stress and protect cartilage from interleukin-1β induced degradation

Abstract

Purpose: Osteoarthritis (OA) is associated with chronic joint inflammation, whereby pro-inflammatory cytokines such as IL-1β upregulate reactive oxygen species (ROS) production while downregulating anti-oxidants in cells. The resulting oxidative stress leads to extracellular matrix degradation, joint inflammation, and chondrocyte death and senescence. The overall goal of this research is to develop a bioactive nanoparticle (NP) system that scavenges ROS in cartilage to modulate the impact of joint inflammation. Manganese dioxide (MnO2) catalyzes the breakdown of hydrogen peroxide (H2O2) and is currently being evaluated for scavenging ROS in inflammatory diseases such as atherosclerosis. We previously reported engineered MnO2 NPs with properties that enable them to penetrate cartilage and become endocytosed by chondrocytes. The objective of this study was to evaluate the chondroprotective mechanisms of MnO2 NPs and their in vivo retention and biocompatibility. Methods: MnO2 NPs were synthesized and characterized as previously described. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2D culture and cartilage explants were confirmed by fluorescent imaging after 24 hours of incubation. Gene expression analysis of both catabolic and ROS mediator genes was conducted on a monolayer of primary bovine chondrocytes challenged by human recombinant IL-1β in the presence of MnO2 NPs after 24-hour culture. Fresh bovine cartilage biopsies were evaluated for loss of glycosaminoglycans (GAG), the production of nitric oxide (NO) and cell viability over a 14-day period when challenged by IL-1β in the presence of MnO2NPs. Articular joint retention of Alexa Fluor 750 and 594 conjugated NPs in Lewis rats was monitored using an in vivo IVIS Imaging System. Long-term cytocompatibility of the particles 6 weeks after intraarticular injection in healthy rat joints was assessed histologically using GEKO©, a tool for quantitative grading of toluidine stained knees. Results: The PEG stabilized MnO2 NPs had a hydrodynamic diameter of 15 nm, TEM size of 8 nm and had a cationic surface charge. The NPs effectively scavenged H2O2, with 5μg/mL MnO2 NPs neutralizing 55% of 100uM H2O2 in PBS. Chondrocytes in monolayer showed uptake of MnO2 NPs without cytotoxicity. Furthermore, the NPs reduced the gene expression of catabolic mediator, heme oxygenase-1, catabolic factor matrix metalloproteinase 3, antioxidant mediator nuclear factor erythroid 2-related factor 2, antioxidants catalase, glutathione peroxidase and manganese superoxide dismutase by chondrocytes (Fig. 1A) indicating protection of the particles against oxidative stress. When incubated with cartilage explants, the MnO2 NPs penetrated through the cartilage depth and were found in the extracellular matrix as well as intracellularly in chondrocytes. In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased GAG loss by 50% and NO production by 40% compared to controls (Fig. 1B and C). Importantly, treatment with MnO2 NPs improved viability of chondrocytes in cartilage explants over the 2-week study. Upon in vivo injection, the MnO2 NPs persisted in the joint space over one week, with 20% of the initial fluorescent signal still observed by day 13 (Fig. 2A and B). Biodistribution and histological analysis (Fig. 2C) revealed accumulation of particles at the chondral surfaces, and colocalization of the NPs with the lacunae of chondrocytes, indicating particle penetration into the cartilage and uptake by chondrocytes following intraarticular injection. A gradient of NPs existed in cartilage with the highest levels of NPs on the articular surface which decreased with tissue depth. Histological analysis also revealed no adverse effects of intra-articular MnO2 NP injection into healthy animals. Histological parameters such as synovium thickness, total cartilage degeneration width and cartilage matrix loss width were comparable in knees injected with the NPs and those injected with saline after 6 weeks, indicating biocompatibility of the particles in vivo (Fig. 2D). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. In vitro, the NPs protected chondrocytes in cartilage explants from IL-1β mediated oxidative stress and apoptosis. Further analysis of the joint confirmed penetration of MnO2 NPs into cartilage in vivo. Given their joint retention time and ROS scavenging capacity, these NPs could target oxidative stress to treat or prevent OA. As the NPs show intracellular localization in chondrocytes, they could also deliver other chondroprotective agents including nucleic acids to target multiple pathways in the OA pathology. Further studies will focus on the therapeutic impact of MnO2 NPs in OA disease models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)