Most neuroprotective drugs had nutrition, repair and protection functions to the central nervous system (CNS), but the entry of drugs was limited by the blood-brain barrier (BBB) which formed by the endothelial cells (Dahl et al., 2018; Fattakhov et al., 2022; Henrich-Noack et al., 2019; Mitusova et al., 2022; Pardridge, 1998). Therefore, it's important to promote the delivery of central nervous system drugs at effective concentrations through BBB to brain injury (Albeck et al., 1997; Datla et al., 2001; Pillai et al., 2013).

It has been proved that amino acids (AAs) may also promote neural plasticity and improved recovery after stroke (Carretero et al., 2023; King et al., 2023; Madsen and Liu, 2023; Morales et al., 2022; Park et al., 2022; Shen et al., 2023; Strasser et al., 1994). AAs are the basic components of protein which is a key nutrient for survival, and the balance of AAs is crucial for the protein synthesis of patients who are unable to absorb AAs. Therefore, it is particularly important to choose safe, nutritious, and economical clinical drugs reasonably. The emergence and development of composite amino acid injection (CAA) provide possibilities for patients. CAA is generally composed of various amino acids, such as glycine (Gly), proline (Pro), serine, and cysteine. At present, amino acid preparations have been widely used as CAA in clinical practice for the treatment of specific diseases or nutritional support (L. J. Gu et al., 2019; Qiu et al., 2021; Shi et al., 2022; Wang et al., 2006; Yue et al., 2013). However, there is currently no effective CAA treatment for brain diseases in clinical practice. Due to the strict selective absorption of AAs by the blood-brain barrier, some AAs that provide neuroprotection are difficult to enter the brain (Nacz, 2016; Takanaga et al., 2002; Zaragozá, 2020). Therefore, the main challenge currently faced is to assist these AAs in safely and effectively entering the central nervous system through the blood-brain barrier.

Our previous studies have proved that multiple amino acids have neuroprotective effect (J Chen et al., 2017; J. Chen et al., 2016; Cheng et al., 2020; Gao et al., 2022; Liu et al., 2019). For example, glycine protects against ischemic neuronal injury through regulating Akt activation by the non-ionotropic activity of NMDARs in stroke animals (J Chen et al., 2017); proline improves the neural functional via ANX6/β1 integrin/Akt signal transduction pathway (Gao et al., 2022); arginine could provide neuroprotection of ischemia injury through suppressing HIF-1 alpha/LDHA-mediated inflammatory response; l-lysine could confer neuroprotection by suppressing inflammatory response via microRNA-575/PTEN signaling after mouse intracerebral hemorrhage injury (Cheng et al., 2020). However, the uptake of these neuroprotective amino acids by BBB is strictly selective. Proline, glycine, alanine, cysteine, glutamic acid and aspartic acid are difficult to enter the brain. At present, chitosan nanoparticles have been widely used in the treatment of brain diseases due to their brain targeting and controlled release properties (Ahmad et al., 2018; Ahmad et al., 2016; Capel et al., 2018; Ding et al., 2016; Gonzalez et al., 2021; J. Gu et al., 2017; Saini et al., 2021; Sava et al., 2020). In our previous study, we have proved that the chitosan nanoparticles (NPs) could efficiently delivered proline into the brain through BBB (Gao et al., 2022).

The purpose of this study was to further test this system for delivering multiple amino acids through BBB to explore improved treatment options for cerebral ischemia. In this study, we prepared two kinds of amino acid-loaded chitosan nanoparticles (AA-NPs) using the ion cross-linking method. This study ‌aimed‌ to develop chitosan nanoparticles capable of loading amino acids for BBB penetration‌ and assess the in vivo distribution of amino acids in the brain after intravenous injection to determine whether AA-NPs penetrated the brain. The therapeutic potential of the nanoparticles was evaluated on the cell-based assays and animal models. The results demonstrated that AA-NPs could cross the BBB and effectively transport multiple amino acids into the brain, thereby reducing ischemic brain injury in rats. Thus, AA-NPs represent an efficient and promising delivery strategy for mitigating‌ ischemic stroke.