Dual‐Functionalized Extracellular Vesicles Promote Brain Repair and Remodeling Following Ischemic Stroke in Mice

Ischemic stroke remains a leading cause of long-term disability and mortality worldwide, with few effective treatment options. A key challenge in recovery is the brain's limited capacity to regenerate neurovascular structures after injury. To address this, we developed a dual-functionalized extracellular vesicle (EV) platform designed to enhance both targeting specificity and therapeutic efficacy for post-stroke repair. Neural stem cell-derived EVs were bioengineered via bio-click chemistry to display RGD peptides, enabling selective binding to integrin αVβ3, which is upregulated on activated endothelial cells in ischemic regions. EVs were concurrently loaded with vascular endothelial growth factor (VEGF), a pro-angiogenic and neurogenic cytokine that also enhances αVβ3 expression-thus creating a synergistic positive feedback mechanism to amplify targeting and tissue repair. Engineered EVs retained normal morphology and showed a 5.2-fold increase in endothelial uptake compared to naïve EVs (p < 0.01). In vitro, they significantly enhanced endothelial cell migration by 2.1-fold (p < 0.05). In a mouse model of transient middle cerebral artery occlusion (tMCAO), intravenously delivered dual-functionalized EVs preferentially accumulated in the ischemic hemisphere, reduced infarct volume by 52.4%, and improved motor coordination (rotarod latency) by 71.8% compared to PBS-treated controls (p < 0.05). Immunostaining revealed enhanced CD31+ microvessel density and increased Nestin+ neural stem and progenitor cell presence, indicating promotion of both angiogenesis and neurogenesis. This study presents a dual-functionalized EV system that combines targeted delivery with therapeutic reinforcement through VEGF loading, offering a potent and synergistic approach for ischemic stroke repair. These findings support further translational development of engineered EVs for neurovascular regeneration.