Proline-Loaded Chitosan Nanoparticles Penetrate the Blood–Brain Barrier to Confer Neuroprotection in Mice Cerebral Ischemia Injury

Proline, a nonessential amino acid, has been demonstrated to possess neuroprotective capabilities in the central nervous system (CNS). However, the mechanisms through which it impacts cerebral ischemia–reperfusion (I/R) injuries remain unclear. Our study observed a decrease in proline concentrations within the ischemic brain tissues of I/R mice. Supplementing proline protected against ischemic neuronal death, both in vivo and in vitro, suggesting its potential as a neuroprotectant. However, our data indicated that proline has a low permeability to the blood–brain barrier (BBB) in mice. Therefore, it is clinically infeasible to develop proline as a stroke therapy. To develop an approach to deliver proline into the injured brain, we engineered a method to transport proline to the injured brain. We prepared chitosan nanoparticles (NPs) loaded with proline utilizing the ion cross-linking method. Our findings demonstrated that the proline-loaded chitosan NPs efficiently passed through the BBB and delivered proline into the injured brain, thereby conferring neuroprotection. Further exploration revealed that proline upregulated the level of annexin A6 (ANX6)/β1 integrin, which, in turn, increased the phosphorylation of cell-survival-promoting kinase Akt. This sequence of events consequently promotes neuronal survival following I/R. In conclusion, our study has developed a neuroprotection approach by which low-BBB-permeable proline is delivered by chitosan NPs into the brain. Furthermore, this study suggests that the neuroprotective role of proline in I/R is mediated by the ANX6/β1 integrin/Akt signaling pathway.