Targeted Delivery of Mdivi-1 via RVG-Functionalized Extracellular Vesicles for Mitochondrial Protection in Ischemic Stroke

Mitochondrial dysfunction is a major contributor to neuronal death following ischemic stroke, promoting excessive reactive oxygen species (ROS) production, inflammation and apoptotic signaling that exacerbate secondary brain injury. Mdivi-1 has demonstrated mitochondrial and neuroprotective potential; however, its clinical translation is hindered by insufficient accumulation in the ischemic brain, higher systemic doses and associated off-target toxicity. This study aimed to develop a neuron-targeting extracellular vesicle (EV)-based delivery system to enhance the targeted delivery of Mdivi-1 to the ischemic brain.

EVs were functionalized with rabies virus glycoprotein peptide (RVG29) via bio-orthogonal click chemistry to generate neuron-targeting RVG-EVs. Mdivi-1 was subsequently loaded into RVG-EVs, and the formulation was characterized for particle size, stability, and drug loading efficiency. HT22 cells were cultured in vitro under oxygen-glucose deprivation (OGD) conditions to evaluate RVG-EV-Mdivi-1 therapeutic potential. For in vivo studies, a mouse transient middle cerebral artery occlusion (tMCAO) model was used to examine the biodistribution, infarct volume, neurological outcomes, mitochondrial dysfunction, oxidative stress, and apoptosis-related signaling pathways.

RVG-EVs demonstrated enhanced neuronal uptake, and under OGD conditions, RVG-EV-Mdivi-1 significantly attenuated mitochondrial fragmentation and ROS accumulation, thereby preserving mitochondrial function and the cellular bioenergetic balance. In tMCAO mice, biodistribution analysis of fluorescently labeled EVs demonstrated enhanced brain accumulation and neuron targeting of RVG functionalized EVs and RVG-EVs-Mdivi-1 compared to naïve EVs. Systemic administration of RVG-EV-Mdivi-1 produced substantial neuroprotective effects at a lower administered Mdivi-1 dose than free Mdivi-1, reducing infarct volume and improving neurological outcomes. Moreover, RVG-EV-Mdivi-1 alleviated ischemic injury and modulated mitochondrial and apoptosis-related pathways, as evidenced by reduced Drp1 and Bax expression and restored Bcl-2 levels.

This study demonstrates that RVG-functionalized EVs offer a promising strategy for targeted delivery of Mdivi-1, enhancing brain accumulation and neuronal uptake while alleviating mitochondrial oxidative damage and neuronal injury following ischemic stroke. These findings support the potential of RVG-EV-mediated Mdivi-1 delivery as a neuron-targeting and dose-sparing therapeutic strategy for ischemic stroke.