Therapeutic Effects of N-Acetylcysteine-Primed, Iron Oxide Nanoparticle-Enhanced Mesenchymal Stem Cell Exosomes in Ototoxicity Hearing Loss.

BACKGROUND: Sensorineural hearing loss caused by ototoxic agents remains irreversible due to the limited regenerative capacity of cochlear hair cells. Exosome-based therapies derived from mesenchymal stem cells (MSCs) offer a promising, cell-free alternative to protect auditory structures by modulating oxidative stress and inflammation. In this study, we evaluated the therapeutic potential of exosomes isolated from nanoparticle (NP) labeled, N-acetylcysteine primed tonsil-derived mesenchymal stem cells (T-MSCs), hereafter referred to as SPISOME-NAC, in kanamycin-induced ototoxicity models. METHODS: T-MSCs were labeled with positively charged PLGA-PEI clustered SPIONs, with or without NAC pretreatment. Antioxidant enzyme activity (SOD, CAT, GSH), ROS levels, and PRDX1 expression were assessed in vitro. Exosomes were isolated and analyzed via nanoparticle tracking analysis. Their therapeutic efficacy was evaluated in both ex vivo cochlear explants and mouse model of kanamycin-induced ototoxicity. Hair cell survival was quantified via Myosin VIIa immunostaining, and auditory function was assessed using auditory brainstem responses (ABR). Pro-inflammatory cytokines (TNF-α, IL-1, IL-6) were measured via qRT-PCR. RESULTS: NAC pretreatment significantly enhanced cell viability, increased GSH activity, and reduced intracellular ROS and PRDX1 levels in NP-labeled T-MSCs. Exosomes derived from NAC-pretreated cells (SPISOME-NAC) conferred superior protection to cochlear hair cells, particularly in the basal turn, and significantly improved hearing thresholds in vivo. Furthermore, SPISOME-NAC treatment downregulated inflammatory cytokines in cochlear tissue. CONCLUSION: SPISOME-NAC exhibit enhanced antioxidant and anti-inflammatory properties, providing functional protection in an ototoxicity-induced hearing loss model. By preventing ROS-mediated mitochondrial damage and apoptosis in cochlear hair cells, NAC interrupts a key pathogenic mechanism in ototoxicity, preserving auditory structure and function. These findings support NAC-primed exosomes as a novel therapeutic strategy for sensorineural hearing loss.