Anhui Province Key Laboratory of Pharmaceutical Preparation Technology and Application, Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM, Anhui University of Chinese Medicine, Hefei 230012, Anhui, China; Engineering Technology Research Center of Modernized Pharmaceutics, Anhui Education Department (AUCM), Hefei 230012, Anhui, China; School of Pharmacy, Institute of Pharmacokinetics, Anhui University of Chinese Medicine, Hefei 230012, Anhui, China; Anhui Genuine Chinese Medicinal Materials Quality Improvement Collaborative Innovation Center, Hefei 230012, Anhui, China; Anhui Academy of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, China.
Int J Pharm. 2024 Mar 5;652:123854. doi: 10.1016/j.ijpharm.2024.123854. Epub 2024 Jan 26.
Borneol is an aromatic traditional Chinese medicine that can improve the permeability of the blood-brain barrier (BBB), enter the brain, and promote the brain tissue distribution of many other drugs. In our previous study, borneol-metformin hydrochloride water/oil/water composite submicron emulsion (B-Met-W/O/W SE) was prepared using borneol and SE to promote BBB penetration, which significantly increased the brain distribution of Met. However, the dynamic images, transport pathway (uptake and efflux), promotion of BBB permeability, and mechanisms of B-Met-W/O/W SE before and after entering cells have not been clarified. In this study, rhodamine B and coumarin-6 were selected as water-soluble and oil-soluble fluorescent probes to prepare B-Met-W/O/W dual-fluorescent SE (B-Met-W/O/W DFSE) with concentric circle imaging. B-Met-W/O/W SE can be well taken up by brain microvascular endothelial cells (BMECs). The addition of three inhibitors (chlorpromazine hydrochloride, methyl-β-cyclodextrin, and amiloride hydrochloride) indicated that its main pathway may be clathrin-mediated and fossa protein-mediated endocytosis. Meanwhile, B-Met-W/O/W SE was obviously shown to inhibit the efflux of BMECs. Next, BMECs were cultured in the Transwell chamber to establish a BBB model, and Western blot was employed to detect the protein expressions of Occludin, Zona Occludens 1 (ZO-1), and p-glycoprotein (P-gp) after B-Met-W/O/W SE treatment. The results showed that B-Met-W/O/W SE significantly down-regulated the expression of Occludin, ZO-1, and P-gp, which increased the permeability of BBB, promoted drug entry into the brain through BBB, and inhibited BBB efflux. Furthermore, 11 differentially expressed genes (DEGs) and 7 related signaling pathways in BMECs treated with B-W/O/W SE were detected by transcriptome sequencing and verified by quantitative real-time polymerase chain reaction (qRT-PCR). These results provide a scientific experimental basis for the dynamic monitoring, transmembrane transport mode, and permeation-promoting mechanism of B-Met-W/O/W SE as a new brain-targeting drug delivery system.
龙脑是一种芳香的中药，能提高血脑屏障（BBB）的通透性，进入大脑，并促进许多其他药物向脑组织分布。在我们之前的研究中，使用龙脑和 SE 制备了龙脑-盐酸二甲双胍水/油/水复合亚微乳（B-Met-W/O/W SE），以促进 BBB 穿透，显著增加了 Met 的脑分布。然而，其进入细胞前后的动态图像、转运途径（摄取和外排）、促进 BBB 通透性的机制尚未阐明。在这项研究中，选择罗丹明 B 和香豆素-6 作为水溶性和油溶性荧光探针，制备具有同心环成像的 B-Met-W/O/W 双荧光 SE（B-Met-W/O/W DFSE）。B-Met-W/O/W SE 可以被脑微血管内皮细胞（BMECs）很好地摄取。加入三种抑制剂（盐酸氯丙嗪、甲基-β-环糊精和盐酸阿米洛利）表明，其主要途径可能是网格蛋白介导和陷窝蛋白介导的内吞作用。同时，B-Met-W/O/W SE 明显抑制了 BMECs 的外排。接下来，将 BMECs 培养在 Transwell 室中建立 BBB 模型，并用 Western blot 检测 B-Met-W/O/W SE 处理后 Occludin、Zona Occludens 1（ZO-1）和 p-糖蛋白（P-gp）的蛋白表达。结果表明，B-Met-W/O/W SE 显著下调了 Occludin、ZO-1 和 P-gp 的表达，增加了 BBB 的通透性，促进了药物通过 BBB 进入大脑，并抑制了 BBB 的外排。此外，通过转录组测序检测到 B-W/O/W SE 处理后的 BMECs 中有 11 个差异表达基因（DEGs）和 7 个相关信号通路，并通过定量实时聚合酶链反应（qRT-PCR）进行了验证。这些结果为 B-Met-W/O/W SE 作为新型脑靶向药物递送系统的动态监测、跨膜转运模式和渗透促进机制提供了科学的实验依据。
