Engineered MSC-EVs Loaded with BDNF-Enhancing Neuropeptides via a Non-Disruptive Method Enhance Post-Stroke Neuroregeneration via Intranasal Delivery

Abstract Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show potential as neuroregenerative therapies. Incorporating bioactive compounds such as neuropeptides that enhance brain-derived neurotrophic factor (BDNF) expression may amplify their therapeutic potential. We developed a clinical-scale method for loading neuropeptides into MSC-EVs, while preserving their structural integrity and therapeutic functionality. Through scalable 3D bioprocessing, we produced high-purity MSC-EVs and evaluated loading methods for encapsulating neuropeptides and full-length BDNF. EVs were characterized using electron microscopy, nanoparticle tracking analysis, and 3D STORM microscopy. The cellular uptake, distribution, and biological effects of neuropeptide-loaded MSC-EVs were tested in vitro and in vivo. Passive incubation was the optimal loading methodfor maintaining EV integrity while achieving effective neuropeptide encapsulation. Active loading methods destabilized the EV membrane despite higher encapsulation efficiency. Neuropeptide-loaded MSC-EVs crossed the blood-brain barrier and significantly enhanced BDNF expression, neurogenesis, and neuroprotection in vitro, ex vivo, and in vivo. Compared with HEK293-derived EVs, MSC-EVs demonstrated superior regenerative effects. In a photothrombotic stroke model, intranasal administration of neuropeptide-loaded MSC-EVs reduced infarct size, improved neuronal survival, and activated neuroprotective pathways mediated by Cyclic AMP Response Element-Binding protein (CREB) phosphorylation. We established a clinically scalable approach for producing neuropeptide-loaded MSC-EVs with potential as next-generation, targeted neuroregenerative therapies for treating stroke and other neurological disorders. Importantly, the EVs used in this study were produced under clinically applicable conditions and characterized according to the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2023 guidelines.