Small extracellular vesicles loaded with neurotrophin-3 confer multitargeted therapeutic effects in treating spinal cord injury

Neural repair following spinal cord injury (SCI) remains a significant medical challenge, necessitating effective therapeutic strategies. This study integrates mesenchymal stem cell-derived small extracellular vesicles (sEVs) with neurotrophin-3 (NT3) to generate NT3-loaded sEVs (NT3-sEVs) and evaluates their efficacy in acute SCI via intranasal delivery. Incorporation of NT3 significantly enhances sEV-mediated neurite outgrowth in cultured cortical neurons. In a mouse contusion SCI model, intranasal administration of NT3-sEVs over seven consecutive days results in superior functional recovery compared to sEV treatment alone, demonstrated by behavioral assessments, electrophysiological measurements, reduced lesion size, enhanced angiogenesis and axonal regeneration, inhibited glial activation, and preserved synaptic plasticity in lumbar motoneurons. Mechanistically, NT3-sEV administration activates the NT3-TrkC signaling pathway, increasing phosphorylation of downstream targets phospholipase C gamma (PLCγ), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK), and upregulates proteins critical for axonal growth and neural plasticity (e.g., growth associated protein 43 (GAP43), synaptophysin, and microtubule associated protein 2 (MAP2)). Transcriptomic and proteomic analyses identify significant differential expression of genes and proteins involved in neural plasticity and immune modulation. Small RNA sequencing further reveals abundant miRNAs common to both sEVs and NT3-sEVs, likely suppressing neuroinflammatory and neuronal death pathways. Thus, intranasal delivery of NT3-sEVs offers a promising therapeutic strategy for SCI by modulating multiple targeted signaling pathways.