Manganese dioxide nanoparticles mitigate oxidative stress in osteoarthritic cartilage

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 role of oxidative stress in OA etiology makes it a therapeutic target to ameliorate some of the devastating symptoms of the disease. 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 hypothesize that MnO2 NPs will protect cartilage from inflammation-induced oxidative stress. To do this, we have engineered chondroprotective MnO2 NPs with properties that enable them to penetrate into the cartilage. Methods: MnO2 NPs were synthesized by the oxidation of potassium permanganate with polyallyamine hydrochloride and further stabilized with polyethylene glycol (PEG). The NPs were characterized for size and zeta potential in phosphate buffer saline (PBS) by Dynamic Light Scattering (DLS) and polydispersity by transmission electron microscopy (TEM). Scavenging capacity of MnO2 NPs was evaluated by a H2O2 colorimetric detection kit. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2-D culture and cartilage explants were evaluated by fluorescent imaging after 24 hours of incubation. The cytotoxicity of NPs to chondrocytes was tested using the MTS colorimetric assay. Fresh bovine cartilage biopsies were challenged with human recombinant IL-1β, to mimic OA-like cartilage, in the presence of MnO2 NPs and evaluated for loss of glycosaminoglycans (GAG), a key component of cartilage extracellular matrix, and production of nitric oxide (NO), a key reactive nitrogen species (RNS) related to oxidative stress in OA, over a 9-day period. The loss of GAG was quantified using spectrophotometric Dimethylmethylene Blue Assay while NO concentrations were evaluated by the Griess reaction. Articular joint retention of Alexa Fluor 750 conjugated NPs in rats was monitored using an in vivo IVIS Imaging System. Results: The PEG stabilized MnO2 NPs had a hydrodynamic size of 15 nm (number weighted) and TEM size of 8 nm. The NPs had neutral charge. The NPs effectively scavenged H2O2, with 5ug/mL MnO2 NPs neutralizing 55% of 100uM H2O2. Chondrocytes in monolayer showed uptake of MnO2 NPs, which escaped the endosomes and localized perinuclearly without cytotoxicity (Fig. 1). The MnO2 NPs penetrated through the depth of the cartilage biopsies and remained in the tissue matrix. Interestingly, the NPs were also endocytosed by resident chondrocytes (Fig. 2). In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased NO production by 80% and reduced GAG loss by 55% compared to controls (Fig. 3A and B). The MnO2 NPs after in vivo injection in articular joints displayed a linear decline (R2 = 0.933) in retention with time. Quantification of fluorescent signals showed that 78% of the initial amount of NPs remained in joints after 5 days (Fig. 4A and B). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. 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 to joint tissues in vivo.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)