Biocatalytic nanobots: modulating oxidative stress in cellular models of inflammatory bowel disease

Introduction: Inflammatory bowel disease (IBD) is a chronic disorder marked by elevated levels of reactive oxygen species (ROS), which exacerbate local inflammation and lead to intestinal mucosal damage (1). Intelligent nanotechnologies, such as biocatalytic nanobots (NBs), offer targeted strategies to reduce oxidative stress (2). This study investigates a self- propelled NB designed to neutralize extracellular ROS and maintain biocompatibility with colonic cells, paving the way for future integrations of bioactive compounds to address intracellular stress further. Methods: The NB core was fabricated using chitosan via ionic gelation with sodium tripolyphosphate (TPP) as the crosslinking agent. A biotin-avidin coupling system was used to anchor catalase onto the NB surface, promoting the decomposition of hydrogen peroxide (H₂O₂) into oxygen (O₂) and enabling propulsion. Mobility was assessed via fluorescence microscopy in ultrapure water (control) and H₂O₂ solutions (30 μM and 300 μM), with the analysis of the mean square displacement (MSD), mean squared speed (MSS), diffusion coefficient, average distance, trajectory bending, and displacement efficiency. Biocompatibility was evaluated on co-culture of colon cell lines (Caco-2 and HT29-MTX), while extracellular and intracellular ROS levels were quantified via the Pierce Quantitative Peroxide and DCFDA/H2DCFDA assays, respectively. Results: The NB exhibited increased propulsion in H₂O₂ solutions, with the MSD as a function of time, highlighting a significant difference in mobility favoring H₂O₂. The comparison of the MSD’s intersection and slope reinforced the improved mobility under H₂O₂ conditions. The MSS did not indicate differences between H₂O and H₂O₂. There was a noteworthy increase in the diffusion coefficient in H₂O₂. The particles traveled a markedly greater distance in H₂O₂, confirming better directional movement in this medium. The trajectory curvature was not different, suggesting similar randomness in movement patterns across both media. The displacement efficiency was significantly greater in H₂O₂, indicating more direct movement from the origin. These findings confirmed robust movement in ROS-rich environments. The NB displayed no detectable cytotoxicity in co- culture of Caco-2 and HT29-MTX cells, and the reduction of extracellular H₂O₂ correlated with a significant decrease in intracellular ROS. This underscores the indirect impact of the neutralization of extracellular ROS on intracellular oxidative stress. Conclusions and Impact: This self-propelled, biocatalytic NB platform offers a powerful approach to addressing oxidative stress in IBD, operating at the extracellular level while maintaining cell viability. Enhanced propulsion in ROS-rich conditions positions these NBs for site-specific applications in inflamed tissues. Future developments will include bioactive payloads to target intracellular ROS, thereby expanding this technology's therapeutic potential for advanced IBD management. Ongoing work will incorporate additional modules to enhance stability, guide navigation, and broaden therapeutic functionalities.