A 3-n-butylphthalide-based nanoagent modulates PINK1/Parkin-associated mitophagy signaling in ischemia-reperfusion brain injury.

Cerebral ischemia and reperfusion induce profound mitochondrial dysfunction in neurons, characterized by excessive mitochondrial fragmentation and persistent accumulation of damaged organelles, which in turn sustain and amplify oxidative stress and inflammatory signaling. Therefore, restoring mitochondrial quality control by enhancing mitophagy to selectively eliminate dysfunctional mitochondria and maintain energy homeostasis represents a promising strategy for alleviating secondary neuronal injury. Here, we develop a phosphatidylcholine (PC)-based supramolecular self-assembly scaffold co-loaded with curcumin (Cur) and 3-n-butylphthalide (NBP) as therapeutic cargos. Hydrophobic interactions and π-π stacking drove the co-incorporation of both drugs into the PC scaffold, resulting in the formation of a stable supramolecular nanoagent (CNP). In neurons subjected to oxygen-glucose deprivation followed by reoxygenation (OGD/R), CNP significantly enhanced intracellular delivery, reduced reactive oxygen species levels, preserved mitochondrial membrane potential, and restored ATP production. Moreover, CNP modulated PINK1/Parkin-associated mitophagy signaling, reduced the accumulation of TOM20 and p62, and suppressed the production of IL-6 and TNF-α. In a transient middle cerebral artery occlusion and reperfusion mouse model (tMCAO/R), intravenous administration of CNP enhanced brain accumulation, reduced infarct volume, and improved neurological scores. These effects were accompanied by reduced CD86-positive pro-inflammatory microglia and increased CD31-positive vascular structures and TUJ1-positive neuronal signals. Overall, CNP represents a promising dual-drug nanoagent strategy for neuroprotection after ischemia/reperfusion by coupling mitochondrial functional preservation with mitophagy reactivation.