Poster N11 - Intracellular localization and ROS scavenging in bovine chondrocytes with enzyme-mimicking nanomaterial

Introduction: Oxidative stress, the imbalance between reactive oxygen species (ROS) and antioxidant function, is a key contributor to the development and progression of osteoarthritis (OA). Maintaining the balance between generating and neutralizing ROS is critical in managing oxidative stress. This complex system is tightly regulated in healthy joints and becomes dysregulated following traumatic joint injury. Current treatments aimed at protecting against this dysregulation are limited by poor bioavailability and limited retention in the target tissue. Nanozymes are an emerging class of biomaterials that provide antioxidant mimicking functions and may overcome limitations of traditional small molecule therapies. Our group has engineered MnO2 NPs as a ROS-targeting nanozyme that catalyzes the breakdown of H2O2 into water and oxygen. MnO2 NPs were engineered to integrate within cartilage owing to their small size (10-12 nm) and positive zeta potential (+30 mV). ROS production predominantly occurs intra-cellularly which means that nanomaterial localization to the surrounding tissue may not be enough for effective treatment. Evaluating nanoparticle fate is a critical component to advancing joint-specific therapies. Tools to measure changes in oxidative stress are limited in physiologically relevant models due to the complexity in measuring short-lived ROS. The development of an ultra-sensitive genetically encoded probe, HyPer7, has significantly advanced our ability to measure site specific changes in redox status. The objective of this work was to i) characterize the enzymatic functions of MnO2 NPs, ii) determine mechanisms of MnO2 uptake into chondrocytes to understand NP fate, and iii) measure compartment specific ROS scavenging using HyPer7 probes.

Materials and

Methods: MnO2 NPs were synthesized following previously reported methods(1). Briefly, potassium permanganate and poly allylamine hydrochloride were mixed in a redox reaction then stabilized by the addition of PEG (MW 3400) and fluorescently tagged with Alexa 488 or 594. Enzymatic functions of MnO2 NPs (5-50 ug/ml) were evaluated in an acellular model using commercially available kits designed to measure catalase and H2O2 scavenging. 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 culture conditions, exposing 2D and 3D chondrocyte cultures to different temperature environments (4C vs. 37C) to quantify changes in MnO2 (5, 20 ug/ml) uptake profiles via flow cytometry and confocal microscopy. Active uptake mechanisms were evaluated through chemical inhibition of endocytic pathways: amiloride hydrochloride (10uM), chlorpromazine hydrochloride (10uM), and methyl-B cyclodextrin (10 uM). To evaluate ROS scavenging activity, bovine chondrocytes were transfected with plasmids encoding HyPer7, treated with MnO2 NPs (5, 20 ug/ml), then washed and exposed to increasing levels of H2O2 over time (2). Oxidation of the probe by H2O2 results in a shift in its excitation wavelength. HyPer7 plasmids contained localization signals targeting the mitochondrial matrix, mitochondrial intermembrane space, or cytosol, and were used to measure compartment specific H2O2 scavenging. Statistical analysis was conducted on GraphPad PRISM 10.2 and presented as mean +/- standard deviation, statistical comparison of means were conducted via a Dunnett’s test or one-way ANOVA with Tukey’s multiple comparisons tests.

Results, Conclusions, and Discussions: MnO2 NPs are uniquely able to enter chondrocytes and scavenge subcellular ROS in multiple compartments. MnO2 NPs (10 ug/ml) possess catalase mimicking functions (2.23 U/ml) similar to the level of CAT in a healthy adult (FigA). When co-incubated with bovine chondrocytes, MnO2 endocytosis is time and concentration dependent. Within 1 hour of co-incubation at standard culture conditions, 77.27% ± 15.81% of cells contain MnO2 (20 ug/ml). Additionally, 62.01% ± 12.13% cells co-incubated at 4C for 1 hour contain MnO2 indicating that chondrocytes can take up MnO2 via both active and passive transport mechanisms (FigB). Further elucidating mechanisms of uptake, it is evident that active transport is primarily through clathrin-mediated endocytosis (only 26/11% ± 15.87% positive cells) following 1 hour of co-incubation with chlorpromazine hydrochloride, a chemical inhibitor of clathrin mediated endocytosis, and 20 ug/ml MnO2. Clathrin mediated endocytosis is the predominant endocytic pathway in mammalian cells and transports cargo to endosomes with several fates. Inhibition of all endocytic pathways still leaves 26.12% ± 7.82% of cells with MnO2, further supporting the role of passive uptake (FigC). This may benefit MnO2 localization to various locations within the cell. MnO2 within chondrocytes localizes to lysosomes, mitochondria, and the cytosol. Colocalization and function of MnO2 within the mitochondria may be driven by the electrostatic interaction between the cationic MnO2 and the negatively charged mitochondrial matrix. HyPer7 probes targeting the mitochondrial matrix and inner membrane space showed a significant dose dependent decrease of oxidative stress (a decrease in the HyPer7 ratio) following treatment with MnO2 (FigD,E). Similarly, a HyPer7 probe targeting the cytosol measured a decrease in oxidative stress, although not dose dependent (FIgF). These results further confirm colocalization of MnO2 NPs to subcellular compartments and may indicate endosomal escape. This work sheds light on the primary endocytic mechanisms of chondrocytes and the ability of these cells to utilize passive transport to take up small, inorganic nanomaterials. This is also the first use of the ultra-sensitive genetically encoded HyPer7 probes to measure ROS scavenging in chondrocytes. MnO2 NPs scavenge ROS at the site of generation and are a promising therapeutic strategy for treating OA.

Acknowledgements (Optional): (1) Kumar 2019 (10.1016/j.biomaterials.2019.119467)
(2) Pak 2020 (10.1016/j.cmet.2020.02.003)