(B-43) Manganese Dioxide Nanozymes as a Chondroprotective Therapy for Osteoarthritis

Introduction:: Nanozymes, antioxidant-enzyme mimicking nano-scale biomaterials, are an emerging therapeutic strategy for several medical applications including neurological conditions, cancer, and more recently orthopedic diseases. Antioxidant-mimicking therapies are a promising approach for modifying disease states caused by oxidative stress. Osteoarthritis (OA), the leading cause of disability in the US, is propagated by oxidative stress in the joint. Current treatment strategies for OA are limited by poor bioavailability and retention in the joint. Application of a nanozyme biomaterial as a therapeutic strategy for OA may overcome some of these conventional limitations. Our group has reported on manganese dioxide nanoparticles (MnO₂ NPs) as an ROS scavenging therapeutic for OA. The MnO₂ NPs were engineered to localize within cartilage, specifically due to their small size (< 15 nm) and cationic charge (+30 mV), improving upon bioavailability and retention limitations of other therapeutics. They have been shown to integrate with the cartilage extracellular matrix, colocalize with chondrocytes, and provide chondroprotection by mitigating the loss of ECM components (glycosaminoglycan) in cartilage explants and decreasing the production of nitric oxide. These chondroprotective effects may be due to the antioxidant mimicking functions of MnO₂, specifically by catalyzing the breakdown of H₂O₂ into water and oxygen. The objective of this work was to i) characterize the enzymatic functions of MnO₂ NPs, ii) evaluate the use of MnO₂ NPs as a therapy in human chondrocyte and cartilage models, and iii) advance the application of MnO2 into an in vivo model of OA.

Materials and Methods:: MnO₂ NPs were synthesized following previously reported methods. Briefly, potassium permanganate and poly allylamine hydrochloride were mixed in a redox reaction producing base-MnO₂ NPs. The base particles were then stabilized with PEG (MW 3400) and fluorescently tagged with Alexa 488 or 594. Enzymatic functions were evaluated in an acellular model using commercially available kits designed to measure catalase and superoxide dismutase function. Human OA cartilage and chondrocytes were isolated from patients undergoing total knee arthroplasty to evaluate the biological effects of MnO₂ on human cells. Cell viability was measured using an MTS cell metabolism assay with MnO₂ concentrations ranging from 5-20 ug/ml. MnO₂ uptake into chondrocytes was visualized with confocal microscopy and quantified via flow cytometry analysis. Passive and active methods of uptake were measured by modulating the culture conditions, exposing cells to different temperature environments (4C vs. 37C) to quantify changes in MnO₂ uptake profiles. MnO₂ fate in relation to uptake mechanism was evaluated by analyzing nanoparticle properties following exposure to pH environments relative to intracellular compartments. At a larger scale, chondroprotective effects in cartilage explants were measured via GAG loss and NO release from cartilage explants exposed to MnO₂ (20 ug/ml) and IL-1B (10 ng/ml) simultaneously. Lastly, the therapeutic effects of MnO₂ NPs were evaluated in a non-invasive injury model of OA. Following injury, weekly intra-articular injections of MnO₂ NPs (15 mg/ml) were given for four weeks. After four weeks, the joints were evaluated histologically using a modified-Mankin grading scale.

Results, Conclusions, and Discussions:: In an acellular model, MnO₂ mimics physiologically relevant levels of catalase (2.23 U/ml) that are found in a healthy adult (Fig 1a). This indicates that MnO₂ nanozymes may rescue low levels of catalase found in OA patients. The addition of MnO₂ (5-20 ug/ml) had no effect on cell viability when compared to an untreated control and there were no significant differences between donors (n = 8) or sex. MnO₂ localized within the chondrocytes within 1 hour of treatment in standard physiological conditions (37C, 5% CO₂) as measured by flow cytometry. MnO₂ were measurable in chondrocytes at both 1 and 3 hours regardless of culture conditions (4C vs. 37C), with at least 60% of cells expressing MnO₂ in both conditions. Uptake was dose dependent, with increased uptake at 20ug/ml compared to 5 ug/ml. Uptake was quantifiable on confocal microscopy within 3 hours of MnO₂ (20 ug/ml) exposure to chondrocytes. These results indicate that MnO₂ enter chondrocytes through both active and passive transport mechanisms and may be subject to various intracellular fates. When exposed to low pH (5.5) conditions, mimicking lysosomal microenvironments, there was a visible change in MnO₂ appearance. Additionally, size and charge could no longer be measured. This may be due to pH-based degradation and warrants further investigation on nanozyme fate following uptake. More broadly, cartilage explants treated with MnO₂ (20 ug/ml) had decreased NO production (Fig 1c) which is consistent with results from bovine cartilage explants. This indicates that the therapeutic effects of MnO₂ are translatable across species. Weekly injections of MnO₂ (15 mg/ml) following a non-invasive knee injury in a rodent model of OA showed improved cartilage protection compared to a saline control (Fig 1d). Although there was not a statistically significant difference in modified Mankin scores between groups at this early timepoint, the trends suggest that chondroprotective effects may have been initiated and may be more measurable at later stages of the disease. This work showcases the antioxidant functions of MnO2, their ability to counter oxidative stress responses, and their potential for use as a chondroprotective agent to slow the development of OA.

Acknowledgements (Optional): : This research was funded by the National Institute of Health, National Institute on Arthritis and Musculoskeletal and Skin Diseases, Awards R01AR080687 and R01AR071335.

References (Optional): : Kumar, S., Adjei, I. M., Brown, S. B., Liseth, O., & Sharma, B. (2019). Manganese dioxide nanoparticles protect cartilage from inflammation-induced oxidative stress. Biomaterials, 224, 119467. https://doi.org/10.1016/j.biomaterials.2019.119467