Synergistic Neuroprotection of Fe3O4 Nanoparticles Combined with Intermittent Theta Burst Stimulation in Ischemic Stroke via Nrf2-Mediated Ferroptosis Regulation

Ying-Ying Huang,1,2 Li-Hong Gui,2 Wen-Xi Yu,1 Rui Tao,1,2 Zhi-Heng Zhu,2 Jing-Jing Li,2 Yi-Ning Zhao,2,3 Xing-Yu Zhang,1,2 Yi-Zhuo Zhang,1,2 Yue Liu,2,3 Xia Bi1,2 1Graduate School, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 2Department of Rehabilitation medicine, Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai, 201318, People’s Republic of China; 3Department of Sport Rehabilitation, Shanghai University of Sport, Shanghai, 200438, People’s Republic of ChinaCorrespondence: Xia Bi, Email [email protected]: Ischemic stroke (IS) accompanied by reperfusion injury results from a cascade of events, including oxidative stress, inflammation, and neuronal apoptosis. Current therapies are limited by poor blood–brain barrier (BBB) penetration and insufficient targeting efficiency.Objective: This study developed an iTBS–Fe3O4 nanoparticle system to enhance magnetic targeting and explore its potential multi-pathway neuroprotective effects.Methods: We constructed a therapeutic platform integrating intermittent theta burst stimulation (iTBS) with magnetic Fe3O4 nanoparticles. Targeting efficiency to the ischemic penumbra was assessed under magnetic guidance with or without iTBS. Potential mechanisms were evaluated by examining ferroptosis-related signaling (Nrf2/GPX4), mitochondrial membrane potential, and microglial polarization. iTBS was compared with conventional rTMS.Results: iTBS significantly enhanced magnetic targeting, resulting in approximately threefold greater Fe3O4 enrichment in the penumbra compared with magnetic guidance alone. This enhanced accumulation was associated with a possible transient modulation of BBB permeability. The combined iTBS–Fe3O4 treatment produced synergistic neuroprotective effects, which were associated with activation of the Nrf2/GPX4 pathway, reduced ferroptosis-related markers, improvement of mitochondrial function, and modulation of microglial polarization toward an anti-inflammatory phenotype. Under the experimental conditions used, iTBS achieved approximately 35% higher targeting efficiency than rTMS. These effects were accompanied by improved functional recovery in rodent stroke models.Conclusion: The iTBS–Fe3O4 system represents a promising preclinical strategy for enhancing nanoparticle targeting and multi-pathway neuroprotection in ischemic stroke. Further studies are required to clarify the precise mechanisms underlying BBB modulation and to evaluate long-term safety and translational potential.Keywords: ischemic stroke, transcranial magnetic stimulation, nanoparticles, polarization of microglia, ferroptosis