RVG‐Modified BMSCs‐Derived Small Extracellular Vesicles Loaded With miR‐21 Alleviate Neuronal Injury Resulted From Excessive Autophagy via Targeting PTEN/Akt/mTOR Pathway After Cerebral Ischaemia

Ischaemic stroke (IS) leads to tragic disability and high adult mortality, while there are limited therapeutic measures for it. Small extracellular vesicles (sEVs) derived from bone marrow mesenchymal stem cells (BMSCs) have been suggested to have satisfactory therapeutic effects on IS by the delivery of their packed microRNA (miRNA). However, systematically administered naïve sEVs are difficult to cross the blood-brain barrier (BBB) and enter the brain parenchyma; also, the low abundance of sEVs cargo miRNAs restricts the possibility of maximising their regulatory functions. Hence, the brain targeting modification and miRNA delivery strategy are crucial for the optimisation of BMSC-sEVs therapeutic effects. In this study, based on sEVs miRNA-seq and analysis of the RNA-seq datasets for ischaemic stroke patients' samples in the GEO database, we identified that miR-21 sharply drops in the ischaemic brain. By the bioengineered BMSCs with RVG-Lamp2b peptide and miR-21-5p overexpression plasmid, RVG-miR21-sEVs were successfully developed. After characterisation of RVG-miR21-sEVs, it showed that RVG-modified sEVs had a high affinity to neurons, and the administration of RVG-miR21-sEVs displayed superior neurological functional rehabilitation and cerebral infarction reduction in the mouse transient middle cerebral artery occlusion (tMCAo) model. Additionally, RVG-miR21-sEVs treatment suppressed neuron autophagy, mitochondria dysfunction and apoptosis after oxygen-glucose deprivation/re-oxygenation (OGD/R) insult. Furthermore, by using dual-luciferase reporter assay, FISH technique, and miR-21 inhibitor and mimics transfection, we validated that the target of miR-21 is PTEN, and the mechanism investigation showed that miR-21 targets the PTEN/Akt/mTOR pathway to antagonise neuronal injury due to excessive autophagy.