Engineered adipose stem cell-derived extracellular vesicles lead to significant improvement of motor function after ischemic stroke by ameliorating neuroinflammation via miR-221-3p delivery

Adipose stem cell-derived extracellular vesicles (ADSC-EVs) have recently emerged as a promising therapeutic agent for mitigating inflammation following ischemic stroke (IS). However, elucidating precisely how ADSC-EVs exert their effects and engineering functionally enhanced, well-targeted ADSC-EVs remain significant challenges. This study aimed to investigate the therapeutic potential of intranasally delivered ADSC-EVs in IS mice, elucidate the underlying mechanism by which ADSC-EVs suppress microglial and inflammatory responses through miR-221-3p delivery, and develop functionally enhanced EVs engineered with this key therapeutic miRNA (miR-221-3p). To evaluate the therapeutic effects and biosafety, we performed behavioral tests, histological and immunofluorescence staining, and initial safety assessments. For mechanistic investigation, we utilized RNA sequencing, Western blot, and a rescue experiment with a constitutively active IKKβ mutant (IKKβ-CA) to confirm pathway specificity. Engineered EVs overexpressing miR-221-3p were constructed for functional validation. We found that ADSC-EVs efficiently reached the ischemic brain and were internalized by microglia, with higher uptake in the pro-inflammatory M1 state. Treatment significantly reduced infarct volume and improved neurological and motor function. Mechanistic investigations using primary microglia and a microglia-neuron co-culture system revealed that ADSC-EVs inhibited microglial inflammation and neuronal apoptosis, thereby creating a more protective microenvironment. We identified miR-221-3p as a key mediator enriched within ADSC-EVs. This miRNA mediated the observed effects by targeting the IKKβ/NF-κB signaling pathway in microglia, confirmed by rescue experiments with a constitutively active IKKβ mutant. Functional validation using engineered ADSC-EVs overexpressing miR-221-3p confirmed the enhanced efficacy in suppressing M1 polarization and amplifying neuroprotection. Notably, engineered ADSC-EVs conferred protection even at lower doses, and initial safety assessments revealed no toxicity or chronic inflammation. We demonstrated that miR-221-3p delivered by ADSC-EVs suppressed microglial M1 polarization via the IKKβ/NF-κB pathway. This suppression inhibited neuroinflammation and neuronal apoptosis, leading to motor functional recovery after ischemic stroke. Repeated intranasal administration of engineered ADSC-EVs showed no detectable toxicity, chronic inflammation, or tumorigenicity, and the therapeutic benefits on motor function were sustained over time. This study elucidates a detailed mechanistic pathway and proposes a novel, safe, and effective therapeutic strategy using miRNA-enriched engineered EVs for the treatment of ischemic stroke.