Engineered iron oxide superparamagnetic nanoparticles (MNPs) offer targeted cutting- edge diagnostic and therapeutic platforms due to their ability to be guided by an external magnetic field, be functionalized and penetrate cell and tissue barriers. In studying the capacity of MNPs to extend neurite outgrowth in differentiated PC12 cells under magnetic force, we observed MNP-induced de-differentiation, loss of neurites, and cell death with increasing concentration of iron oxide, but not dimercaptosuccinic acid (DMSA) used for MNP coating. Mounting evidence suggests that enhanced reactive area, permeability and resistance to biodegradation of nanoparticles promote their cytotoxic potential relative to molecular or bulk counterparts, implicating oxidative stress (OS) as a key paradigm of nanotoxicity. A 3-tier process, OS manifests in activation of reactive oxygen species (ROS) and antioxidant defense (tier I), pro-inflammatory response (tier II) and DNA damage leading to apoptosis (tier III). Upon their in vivo application, nanoparticles are quickly challenged by macrophages, which both buffer potential nanotoxicity of nanoparticles and reduce circulation time necessary for their therapeutic and diagnostic use. In a series of pilot in vivo studies, we used rat sciatic nerve as a combination model for assessment of direct neurotoxicity and effective intraneuronal macrophage infiltration that is unique to peripheral nerve. Within 48 hours of intrafascicular microinjection of anionic DMSA-coated MNPs (AMNPs) that are highly stable, water soluble and resistant to agglomeration, we observed a robust influx of macrophages, activation of heme oxygenase-1, interleukin-12, matrix metalloproteinase (MMP)-9 and caspase 3, all consistent with the oxidative stress paradigm. In contrast, only mild neurotoxic changes were seen in the corresponding sham procedures, control DMSA and dextran-coated iron oxide MNPs microinjections. Utilizing a combination of engineering and biological in vitro and in vivo approaches, this program aims to determine the mechanisms and target cells of central and peripheral neurotoxicity induced by iron oxide MNPs. Emphasis will be made on studying the role of surface chemistry (DMSA, dextran and gold) on activating oxidative stress signaling and biodistribution in vitro and in vivo. A combination of SQUID magnetometry, light, electron and confocal neuropathology, ROS-mediated pro- inflammatory and pro-apoptotic cell signaling analyses and in vivo sensory and motor behavioral assessments will be used. The overall goal of this proposal is to develop and test the engineering strategies that are safe for MNP use in therapeutic and diagnostic platforms in the nervous system. PUBLIC HEALTH RELEVANCE Magnetic nanoparticles (MNPs) offer cutting-edge drug delivery, molecular imaging and tissue engineering tools for all areas of medicine, including neurosciences. However, their enhanced reactive area, permeability and resistance to biodegradation promote their toxic potential. This proposal aims to determine the mechanisms of MNP neurotoxicity, immune activation of defense macrophage system, and develop advanced MNP formulation that are safe and robust for diagnostic, therapeutic and research use in the nervous system.