## 4 Discussion  
In recent years, targeted nanomedicine has emerged as an effective treatment modality for delivering drugs to the site of cerebral ischemia [39]. Various receptors and transporters are highly expressed on brain capillary endothelial cells to facilitate the transport of nutrients [40]. By modifying drugs with ligands of these receptors or transporters, significant increases in brain drug accumulation were observed. It was demonstrated that integrin \(\alpha\
u\beta\)3 was initially detected in the ischemic area of the brain 2 h after middle cerebral artery occlusion (MCAO) and is highly expressed thereafter [22; 41]. In a transient MCAO mouse model, exosomes modified with c(RGDyK) significantly accumulated in the brain lesion area. Further experiments revealed that the targeting ability of cRGD-Exosome was mediated by the recognition of \(\alpha\
u\beta\)3, which was induced expressing on brain vascular endothelial cells by ischemia [42]. In another study, bLXW7, a ligand of integrin \(\alpha\
u\beta\)3, modified nanoparticle CeNPs exhibited effective accumulation in the peri-infarct brain region, and protected neurons from acute cerebral ischemia-reperfusion injury [43]. These findings suggest that integrin \(\alpha\
u\beta\)3 ligands have inherent advantages in delivering drugs to cerebral ischemic sites. In this study, we designed an \(\alpha\
u\beta\)3 targeting agent LA-1 by conjugating a neuroprotective molecule loganin with one RGD-containing peptide for neuroprotection against cerebral ischemia-reperfusion injury. Our data demonstrated that LA-1 penetrated BEnd.3 cells rapidly through \(\alpha\
u\beta\)3 mediated cellular uptake, leading to the intracellular amount of LA-1 was approximately 500-fold higher than that of loganin after 3 h of incubation. In vivo, although the plasma half-life of LA-1 is shorter than that of loganin, we still detected the dramatically higher brain accumulation level of LA-1. Both in vitro and in vivo results supported our hypothesis that conjugation with \(\alpha\
u\beta\)3 ligands increases active molecules' brain delivery. As the active group of LA-1, loganin was reported to protect neurons from cerebral ischemia-reperfusion injury via reducing oxidative stress and anti-inflammation [19]. The antioxidant activity of LA-1 was validated through free radical scavenging and TBHP-induced ROS generation assays. Compared with loganin, the activity of LA-1 did not significantly increase in inhibiting TBHP-induced ROS generation in BEnd.3 cells. This observation conflicted with the result from the cellular uptake assay, indicating structure modification on the selected site may decrease the antioxidant activity of loganin in vitro. In addition, LA-1 effectively protected BEnd.3 cells from TBHP-induced cell death at the given condition, but not loganin. This finding supported the potential of LA-1 against cerebral ischemia-reperfusion injury and suggested that reducing oxidative stress may not be the only mechanism of cell protection of LA-1. Using the MCAO model, we evaluated the neuroprotective activity of LA-1 against cerebral ischemia--reperfusion injury. Intravenous injection with LA-1 effectively restored cerebral blood flow and significantly improved neurological deficit scores in MCAO rats. TTC staining and pathology sections displayed LA-1 treatment reduced infarct volume, restored the ischemia-reperfusion injury induced morphology changes of the cortical and hippocampal zone, and inhibited neuron apoptosis. These data confirmed the therapeutic potential of LA-1 against cerebral ischemia-reperfusion injury. We propose that the neuroprotective effect of LA-1 is achieved through a dual mechanism. On one hand, LA-1 enters brain endothelial cells via the \(\alpha\
u\beta\)3 integrin receptor, reducing ROS levels within these cells, thereby indirectly protecting the cerebrovascular system and neurons.