Borneol and lactoferrin dual-modified crocetin-loaded nanoliposomes enhance neuroprotection in HT22 cells and brain targeting in mice

Crocetin (CCT), a natural bioactive compound extracted and purified from the traditional Chinese medicinal herb saffron, has been shown to play a role in neurodegenerative diseases, particularly depression. However, due to challenges with solubility, targeting, and bioavailability, formulation development and clinical use of CCT are severely limited. In this study, we used the emulsification-reverse volatilization method to prepare CCT-loaded nanoliposomes (CN). We further developed a borneol (Bor) and lactoferrin (Lf) dual-modified CCT-loaded nanoliposome (BLCN) for brain-targeted delivery of CCT. The results of transmission electron microscope (TEM) and particle size analysis indicated that the size of BLCN (∼140 nm) was suitable for transcellular transport across olfactory axons (∼200 nm), potentially paving a direct path to the brain. Studies on lipid solubility, micropolarity, and hydrophobicity showed that BLCN had a relatively high Lf grafting rate (81.11 ± 1.33 %) and CCT entrapment efficiency (83.60 ± 1.04 %) compared to other liposomes, likely due to Bor improving the lipid solubility of Lf, and the combination promoting the orderly arrangement of liposome membrane molecules. Microplate reader and fluorescence microscopy analysis showed that BLCN efficiently promoted the endocytosis of fluorescent coumarin 6 into HT22 cells with a maximal fluorescence intensity of (13.48 ± 0.80 %), which was significantly higher than that of CCT (5.73 ± 1.17 %) and CN (12.13 ± 1.01 %). BLCN also exhibited sustained function, remaining effective for more than 12 h after reaching a peak at 1 h in cells, while CN showed a significant decrease after 4 h. The uptake mechanisms of BLCN in HT22 cells mainly involve energy-dependent, caveolae-mediated, and microtubule-mediated endocytosis, as well as micropinocytosis. Furthermore, BLCN displayed a significant neuroprotective effect on HT22 cells in glutamate-, corticosterone-, and H