Therapeutic Effects of miR-21-5p-Enriched Milk Extracellular Vesicles on Alzheimer's Disease-Associated Neurotoxicity in Vitro

Progressive neuronal degeneration linked to oxidative stress, mitochondrial malfunction, and persistent neuroinflammation are the hallmarks of Alzheimer's disease (AD). The current study investigated the neuroprotective potential of miR-21-5p-loaded milk-derived small extracellular vesicles (sEV_miR-21-5p) against amyloid beta (A(3)-induced toxicity in SH-SY5Y neuroblastoma cells. Milk-derived sEVs were isolated and characterized following the MISEV 2018 guidelines, and miR-21-5p was actively loaded into the vesicles. A(3-induced oxidative stress was effectively reduced by treatment with sEV_miR-21-5p, as evidenced by decreased levels of ROS, MDA, LDH, and GPX1, along with restored SOD activity. Furthermore, sEV_miR-21-5p mitigated mitochondrial dysfunction, indicated by increased TFAM expression and decreased Cyt-c, PINK1, and DNM1L levels. The treatment also downregulated inflammation-associated signaling molecules (TNF-alpha and ICAM1) while enhancing BDNF expression, suggesting modulation of neuronal stress-response and survival pathways. In addition, miR-21-5p delivery normalized neuronal cytoskeletal and stress-related protein (NfL), preserved synaptic protein expression (CPLX2 and SMOC1), and significantly reduced tau hyperphosphorylation and A(3 accumulation. These findings demonstrate that milk-derived sEVs serve as efficient, biocompatible carriers for miR-21-5p, enabling targeted delivery and functional recovery in neuronal cells. Taken together, this study underscores the therapeutic potential of sEV_miR-21-5p as a biocompatible and scalable platform for targeted intervention in AD.