A multi-target nano-therapy against cerebral ischemia/reperfusion injury via combinatorial inhibition of neuroinflammation and pyroptosis

Background & purpose: ischemic stroke reperfusion injury involves a vicious cycle of neuroinflammation, pyroptosis, and oxidative stress. Single-target therapies have limited efficacy. In this study, we aimed to develop an actively targeted, multi-drug combinatorial nano-platform for coordinated intervention against ischemia/reperfusion (I/R) injury. Methods: we synthesized CX3CL1-functionalized ZIF-8 nanoparticles co-loaded with disulfiram (DFL, a GSDMD-N pore inhibitor), paquinimod (PAQ, a TLR4/NF-κB inhibitor), and siNINJ1 (inhibiting membrane rupture). The nanoparticles were systematically characterized. Their neuroprotective effects and mechanisms were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model and an oxygen-glucose deprivation/reoxygenation (OGD/R) co-culture model in vitro. Results: the nanoparticles exhibited pH-responsive release and active targeting to the ischemic penumbra. In vivo and in vitro results demonstrated that they synergistically inhibited the TLR4/NF-κB/NLRP3 signaling axis and pyroptosis execution (GSDMD, caspase-1), promoted microglial polarization towards the M2 phenotype, reduced pro-inflammatory cytokines (IL-6, TNF-α), and alleviated oxidative stress and neuronal apoptosis, ultimately leading to significantly reduced infarct volume and improved neurological recovery. Conclusion: we successfully developed an “active targeting—multi-drug synergy—cascade intervention” nano-therapeutic platform that effectively mitigates cerebral I/R injury through multi-pathway coordination, offering a novel combinatory strategy for ischemic stroke treatment.