"鼻通脑络"多通路细胞模型组的构建及通窍散瘀共聚物纳米乳鼻脑转运特性研究

Chinese medicine theory of "nasal connect with brain" provide fundamental basis for traditional Chinese medicine treating encephalopathy though nasal, but the nose-brain transportation, involving in many pathway, such as olfactory nerve pathway, systemic circulation pathway and so on, is complex. Current in vitro models can not fully and thoroughly discuss the transport mechanism of drug, especially compound multicomponent, from nose to brain and restrict the research of nasal preparation. This project will establish a series of cell models including nasal mucosa cell and olfactory nerve cell co-culture simulating direct pathway, nasal mucosa cell and MDCK cell co-culture simulating indirect pathway and the pathway in pathological conditions. We make the TongQiaoSanYu as model drugs and prepared nano-emulsions with mPEG-PLA which is quick passing into brain, high drug loading and low irritation. Then combining the models and the results of pharmacokinetics, we'll systematically study the influence and mechanism of traditional Chinese medicine multicomponent, excipient, compatibility, formulation, pathway and pathology conditions. It will provide new researching idea and models for choosing excipient, designing dosage form and evaluating drugs of nasal formulation treating encephalopathy, expand the modern medicine value of "nasal connect with brain" from the point of molecular pharmaceutics theory, and possess prominent innovation of theory and application.

中医"鼻通脑络" 理论已为经鼻给药治疗脑病提供了理论基础，但由于鼻脑转运涉及如嗅黏膜、嗅神经、体循环-BBB等多通路途径，现有各类转运模型尚不能体现药物成分经鼻给药后经多通路入脑的特性，制约了治疗脑病经鼻给药制剂的深入研究。本课题拟通过将嗅黏膜-嗅神经细胞共培养模拟鼻脑直接通路，黏膜-MDCK细胞共培养模拟鼻脑间接通路及培养中风病理条件下鼻脑通路模型，创新性构建符合鼻-脑转运多通路特性的生理-病理细胞模型组。并选用通窍散瘀组分方为模型药，以新型嵌段共聚物制备入脑快、载药高、刺激小的鼻腔纳米制剂，基于上述模型组合，结合微透析体内药动学，系统探讨中药复方的药物因素、辅料因素、剂型因素等对生理、病理多通路经鼻入脑的影响和作用机理；为治疗脑病经鼻给药制剂的辅料筛选、剂型设计、药物评价提供新的研究思路和模型，以现代分子药剂学理论拓展中医"鼻通脑络"的作用机理，具有突出的理论创新和应用价值。

中医“鼻通脑络”理论已为经鼻给药治疗脑病提供了理论基础，但鼻脑转运涉及多通路途径，且现有的模型不能体现药物成分经鼻给药后经多通路入脑的特性。课题组成功建立了鼻-脑间接通路体外模型（Calu-3和MDCK-MDR1共培养）、鼻-脑直接通路体外模型（OECs与Calu-3共培养）、正常和糖氧剥夺下MDCK-MDR1细胞模型；以通窍散瘀方主要成分为模型药物，考察了不同极性成分在上述模型中的转运行为；成功制备了刺激性小、载药量高的通窍散瘀微乳，并基于上述细胞模型组研究了通窍散瘀微乳的吸收、转运特性及机理。结果显示葛根素在Calu-3细胞、鼻-脑间接通路中的转运均为被动扩散；薄荷脑可以促进葛根素的渗透，相关机制与细胞的紧密连接、P糖蛋白、细胞膜流动性、Ca2+-ATP 酶介导的代谢活动有关，而芍药苷则不能促进葛根素的渗透；摄取实验显示，直接通路对纳米乳的摄取随粒径增大而减少；病理状态（糖氧剥夺）与微乳制剂均促进药物跨过血脑屏障进入中枢神经系统，同时微乳亦可进一步促进葛根素在病理状态下BBB的被动转运；通窍散瘀微乳促进葛根素在血脑屏障模型转运的机理与微乳打开细胞间紧密连接，增加细胞膜流动性，降低细胞膜电位，提高细胞内Na+/K+-ATPase、Ca2+- ATPase活性相关；体内药动学实验结果与细胞模型组实验结果之间存在较好的一致性，进一步说明所建立的体外细胞模型能在一定程度上反映药物/制剂体内生物药剂学特性，模型可靠。