ROS-responsive, brain- and M1 microglia-targeting modified ginkgetin-loaded smart liposomes ameliorate cerebral ischemia by HIF-1α-mediated negative regulation of microglia pyroptosis

Ischemic stroke triggers a cascade of mitochondrial dysfunction, oxidative stress, neuroinflammation, and pyroptosis, ultimately leading to neuronal injury and neurological deficits. Therapeutic efficacy is often limited by inadequate blood–brain barrier penetration and off-target effects. To address these challenges, we designed 3R@Lipo/Gink, a biocompatible liposomal formulation modified with an ROS-responsive TK polymer and two functional peptides—RVG29 for enhanced brain delivery and MG1 for microglia enrichment—to enable precise transport of ginkgetin, which has been demonstrated to exert neuroprotective effects through multiple potential mechanisms, to ischemic lesions.In a middle cerebral artery occlusion/reperfusion model, 3R@Lipo/Gink markedly reduced infarct size, alleviated neuronal injury, and improved motor performance. Single-cell RNA sequencing and in vitro co-culture experiments identified microglia and neurons as the primary responsive cell types. Mechanistic studies showed that 3R@Lipo/Gink suppressed HIF-1α expression, thereby downregulating c-myc–mediated microglial proliferation and attenuating NLRP3-dependent pyroptosis. These protective effects were reversed by FG-4592, a prolyl hydroxylase inhibitor that stabilizes HIF-1α, supporting the involvement of the HIF-1α pathway. Through mitigating microglial overactivation and interrupting the inflammatory-pyroptotic loop, 3R@Lipo/Gink ultimately remodels the microglia-mediated inflammatory microenvironment around neurons and improves functional recovery. These findings highlight 3R@Lipo/Gink as a promising targeted nanotherapeutic strategy for ischemic stroke and other nervous system diseases. 3R@Lipo/Gink, surface-modified with brain-targeted RVG29 peptide and microglial cell-targeted MG1 peptide and loaded with GK, was injected via the tail vein and accumulated in the ischemic brain. After the MG1 peptide binds to the M1-type microglia in the microenvironment with ROS enrichment, it triggers TK-induced liposome disintegration and drug release. The released GK exerts various therapeutic effects, such as inhibiting oxidative stress, microglial pyroptosis, and proliferative activation.