不同药性芳香药促进不同机械强度纳米制剂经鼻入脑的多尺度研究

Chinese medicine theory of “nasal connect to brain” and the confirmation of nose-brain pathways from anatomy have provided sufficient basis for the development of intranasal preparations for brain-targeting. Studies have shown that the drug exposure in brain was increased when the nano-preparations were administrated intranasally combined with aromatic chinese medicines. The transportation mechanism of different types of nano-preparations (flexible- and rigid-type) from nose to brain and the penetration-enhancing mechanism of aromatic drugs are valuable for the development of intranasal drug delivery system for brain-targeting based on the combination of different technologies..Based on the viewpoints above, an in vitro model of nasal-brain pathways (olfactory nerve pathway and nasal mucosa-BBB pathway) will be set up. The transportation and cellular uptake behavior of nose-brain models, the biophysical properties of cells and nano-preparations, and the intercellular junction of membrane barriers will be studied to investigate the influence of aromatic chinese medicines with different property (warm and cool) on nano-preparations and cells in macroscopic and mesoscopic scales. The enhancement mechanism of aromatic chinese medicines in the transportation of different nanocarriers will be discussed in the perspectives of biopharmaceutics, biophysics and cytobiology to provide strong evidences to the superiority of the combination of pharmaceutical and chemical permeation technologies in the intranasal drug delivery system. Furthermore, our work will bring the advantages of Traditional Chinese Medicine (TCM) and modern medicine in the treatment of encephalopathy together and show a new way to integrate TCM with modern medicine.

中医“鼻窍通脑”理论及现代医学对鼻脑通路的确证已为经鼻脑靶向递药系统的开发提供了充分的依据。研究表明，芳香开窍药与纳米制剂联用后经鼻递药能够进一步提高入脑药量。探索不同类型纳米制剂（柔性、刚性）的经鼻入脑转运机理及不同药性芳香药对纳米制剂鼻脑转运的促进机理，对于开发基于多技术联用的新型鼻腔递药系统具有重要价值。.基于上述认识，本课题拟构建鼻脑通路（嗅神经通路，鼻黏膜-BBB通路）体外细胞模型，在此基础上，在宏观及介观尺度下，通过比较配伍温、凉药性芳香药前后不同类型纳米制剂跨膜转运和细胞摄取行为的变化、细胞表面物理性质的变化、胞吞途径和胞内分布的变化、膜屏障细胞间连接的变化，从生物药剂学、生物物理学、细胞生物学角度揭示温、凉药性芳香药促进纳米制剂入脑的机理，为化学和药剂学促透技术联用提供实验依据。同时，为融合传统医学与现代医学在治疗脑病方面的优势，探索中西医结合的新途径，提供新的思路。

本课题从基于传统中医药理论及现代纳米技术研究经鼻脑靶向递药系统出发。采用乳化-溶剂挥发法制备FITC标记的PLGA-NPs，并与介孔二氧化硅（MSNs）同期展开实验，采用透射电镜（TEM）、马尔文粒度仪和原子力显微镜（AFM）对两种纳米载体的物理性质进行表征，结果表明PLGA-NPs刚性小于MSNs，为考察刚性是否会对纳米载体鼻脑转运过程中的行为存在影响，同时探索配伍芳香药薄荷醇或莪术醇对纳米载体鼻脑转运行为及机理的影响，首先以新生大鼠为取材对象，经流式分选纯化构建鼻脑直接通路-嗅神经鞘细胞模型（OECs），其次以小鼠脑微血管内皮细胞（bEnd.3）模拟鼻脑间接通路血脑屏障部分。采用多功能酶标仪及流式细胞仪，探索刚性及配伍因素对两种纳米粒鼻脑转运的行为学影响，结果显示①MSNs更易于被bEnd.3及OECs细胞摄取，而PLGA-NPs更易于跨bEnd.3细胞单层转运。②配伍芳香药后，两种纳米载体在摄取速度及程度上均出现差异，提示不同芳香药对两种纳米载体摄取行为存在不同程度的影响；跨膜转运时，配伍芳香药均对PLGA-NPs渗透性能具有增强作用，而对MSNs的渗透性能未产生显著影响。在研究纳米载体鼻脑转运的机理时，①采用AFM考察两种纳米载体配伍芳香药对bEnd.3及OECs细胞膜表面物理性质的影响，结果表明芳香药配伍会改变细胞膜表面物理性质。②采用荧光漂白恢复技术探讨芳香药对bEnd.3细胞膜流动性的影响，结果表明PLGA-NPs配伍后膜流动性增大，而MSNs仅在配伍莪术醇时才表现出更高的膜流动性。③通过对比抑制剂作用前后细胞内相对荧光值计算胞吞通路抑制率来确定配伍因素对潜在摄取路径的影响，结果表明芳香药物会影响纳米制剂的摄取通路。④为进一步观察细胞内摄取情况，通过激光共聚焦显微镜成像观察两种纳米载体及其配伍的溶酶体共定位情况，结果显示配伍薄荷醇或莪术醇均可能促进PLGA-NPs部分逃逸溶酶体进而在脑内传递，而配伍作用对MSNs则没有显著影响。⑤激光共聚焦显微镜成像结果显示，配伍芳香药后会对紧密连接蛋白产生不同程度的影响，从而可能打开细胞间紧密连接。