Abstract：
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 H2O2-induced models. Tissue fluorescence image analysis of mice showed that BLCN exhibited substantial retention of fluorescent DiR in the brain after nasal administration for 12 h. These findings suggest that CCT has the potential for cellular uptake, neuroprotection, and targeted delivery to the brain following intranasal administration when encapsulated in Bor and Lf dual-modified nanoliposomes.
摘要：
藏红花素(CCT),一种从中草药藏红花中提取和纯化的天然生物活性化合物，已被证明在神经退行性疾病中起作用，尤其是抑郁症。然而,由于溶解度的挑战，瞄准,和生物利用度，CCT的制剂开发和临床使用受到严重限制。在这项研究中,我们使用乳化-反向挥发法制备了负载CCT的纳米脂质体（CN）。我们进一步开发了冰片（Bor）和乳铁蛋白（Lf）双重修饰的CCT负载纳米脂质体（BLCN），用于脑靶向递送CCT。透射电子显微镜（TEM）和颗粒尺寸分析的结果表明，BLCN的尺寸（〜140nm）适合跨嗅觉轴突（〜200nm）的跨细胞转运，可能为大脑铺路.脂溶性研究，微极性,疏水性表明BLCN与其他脂质体相比具有较高的Lf接枝率(81.11±1.33%)和CCT包封率(83.60±1.04%),可能是由于Bor改善了Lf的脂溶性，和组合促进脂质体膜分子的有序排列。酶标仪和荧光显微镜分析表明，BLCN有效促进荧光香豆素6向HT22细胞的内吞作用，最大荧光强度为(13.48±0.80%)。显著高于CCT(5.73±1.17%)和CN(12.13±1.01%)。BLCN还表现出持续的功能，在细胞中达到1小时的峰值后，保持有效超过12小时，而CN在4h后显示显着降低。BLCN在HT22细胞中的摄取机制主要涉及能量依赖性，Caveolae介导的，和微管介导的内吞作用，以及微胞吞作用。此外，BLCN对谷氨酸中的HT22细胞显示出显着的神经保护作用-，皮质类固醇-,和H2O2诱导的模型。小鼠的组织荧光图像分析表明，BLCN在鼻腔给药后12小时表现出荧光DiR在大脑中的大量保留。这些发现表明CCT具有细胞摄取的潜力，神经保护,以及当封装在Bor和Lf双重修饰的纳米脂质体中时,在鼻内施用后靶向递送至脑。
