Evaluating Intracellular Location and ROS Scavenging by Manganese Dioxide Nanoparticles in Chondrocytes

Oxidative stress, the imbalance between reactive oxygen species (ROS) and antioxidant function, is a key contributor to the development and progression of osteoarthritis (OA). ROS-scavenging nanomaterials, which overcome stability and bioavailability limitations of conventional antioxidants, are an emerging class of potential therapies for osteoarthritis. Previously, we reported on engineering manganese dioxide nanoparticles (MnO₂), which catalyze the breakdown of hydrogen peroxide (H₂O₂), and have properties favorable for cartilage localization and subsequent chondroprotection against inflammation. The objective of this study was to determine the mechanisms of MnO₂ uptake, intracellular fate, and ROS scavenging functions in chondrocytes at the subcellular level. Uptake mechanisms were evaluated in bovine chondrocytes based on temperature dependence and selective chemical inhibition of endocytic pathways. The overall ROS scavenging functions of the MnO₂ were determined in both acellular and cellular systems. To determine compartment-specific ROS scavenging, genetically encoded H₂O₂ sensors (HyPer7) were employed to detect H₂O₂ in the mitochondria and cytosol of chondrocytes with and without MnO₂ treatment. MnO₂ produced catalase-like effects and decreased global levels of oxidative stress in chondrocytes exposed to exogenous H₂O₂. When coincubated with bovine chondrocytes at 4 or 37 °C, MnO₂ leveraged both energy-dependent and independent uptake mechanisms to enter cells. Once inside the cells, MnO₂ scavenged H₂O₂ in the mitochondrial matrix, mitochondrial inner membrane space, and cytosol, corresponding to MnO₂ localization in the mitochondria. Importantly, colocalization of MnO₂ with mitochondria did not adversely impact mitochondrial respiration in healthy chondrocytes. This study is the first to utilize HyPer7 probes to measure ROS scavenging in chondrocytes, which could be a promising strategy for interrogating oxidative stress mechanisms in OA. This study provides mechanistic insights into how MnO₂ localize within chondrocytes and scavenge H₂O₂ in compartment-specific subcellular regions, thereby advancing a promising strategy for mitigating oxidative stress in chondrocytes.