肠上皮细胞转运体介导的口服纳米载体的构建及其胞内转运的分子调控机制

The transmembrane transport efficiency of plain polymeric micelles has been not satisfied, and the intracellular trafficking route and molecular regulation mechanism for polymeric micelles lacked of systematic and deep research. To this end, according to the characteristic of small intestine, the new idea has been developed to effectively enhance the transmembrane transport efficiency of polymeric micelles in the present research based on the targeting strategy. The Phe-Gly, the substrate of intestinal peptide transporter 1 (PETP1), modified polymeric micelles are to construct based on PEG-PLA. The appropriate composition of PLA-PEG-Phe-Gly is determined according to the affinity of PLA-PEG-Phe-Gly to PETP1 by calculation with molecular dynamics simulation and competitive inhibition test, the stability in intestine and in vitro absorption. The optimal content of PLA-PEG-Phe-Gly in micelles is determined by transmembrane transport of polymeric micelles across Caco-2 cell monolayer. Using advanced biological technologies and methods, the co-localizations of micelles with cell organelles are observed by using CLSM to elucidate the intracellular trafficking route for polymeric micelles, which is further clarified by the determination of the proteins adsorbed by micelles using proteomics methods in combination with LC/MS/MS. The expression of intracellular specific proteins is down-regulated or inhibited by using knockdown experiments with siRNAs. The molecular regulation mechanism of intracellular trafficking for polymeric micelles is revealed by investigation of the effect of the associated proteins on the transmembrane transport efficiency of polymeric micelles. The results of this research might provide the theoretical base and guidance for construction of nanocarriers with low toxic, highly selective and efficient transmembrane transport.

普通聚合物胶束的跨膜转运效能不尽如人意，对其胞内转运途径及分子调控机制的研究尚缺乏系统性和深度。本项目针对小肠特点，基于靶向策略提出有效提高胶束跨膜转运效能的新思路，拟构建寡肽转运体PEPT1的底物Phe-Gly修饰的PEG-PLA胶束。依据分子动力学模拟和竞争性抑制等确定的PLA-PEG-Phe-Gly与PEPT1的亲和性、稳定性和体外吸收等确定合适组成的PLA-PEG-Phe-Gly；根据胶束跨Caco-2细胞单层转运确定胶束中PLA-PEG-Phe-Gly的最佳含量；用先进的生物学方法，借助共聚焦显微镜观察胶束与细胞器的共定位，并用蛋白质组学联合LC/MS/MS确定胶束吸附的蛋白，阐明胶束的胞内转运途径；用siRNA下调/抑制胞内相关转运蛋白的表达，研究其对胶束跨膜转运之影响，从分子水平上揭示其胞内转运的机制。本研究将为构建低毒、高选择性和高跨膜转运效能的纳米载体提供理论依据和指导。

提高难溶性药物水溶性和透膜性是改善其口服吸收的主要策略，其中普通聚合物胶束能在一定程度上克服影响药物吸收的某些不利因素，这可能归因于胶束的增溶、并携载药物跨膜转运、保护药物免受分解代谢酶的降解以及外排转运系统的外排等。虽然聚合物胶束成千上万倍提高了难溶性药物的溶解度，但药物的跨膜转运效能却只提高了几倍至十几倍，药物的口服吸收仍不尽如人意。因此，如何针对小肠上皮的特点，进一步有效提高聚合物胶束的跨膜转运效能，进而大幅提高难溶性药物的口服吸收是当前亟待解决的重大难题。另外，目前对于口服聚合物胶束跨膜转运机制的研究仅停留在入胞和囊泡水平，极少涉及其胞内的转运途径，更没有深入到真正决定胶束转运命运的蛋白质分子水平。本项目基于靶向策略提出有效提高口服纳米载体系统跨膜转运效能的新思路，对上述重要问题进行了深入研究，获得了一系列的研究成果：基于寡肽转运体成功构建了高选择性和高跨膜转运效能的口服聚合物胶束纳米载体；寡肽转运体底物的修饰改变了聚合物胶束的跨膜转运途径，主要通过网格蛋白/脂筏介导；寡肽转运体介导的聚合物胶束在小肠上皮细胞内的转运途径为内涵体-内质网-高尔基体，小窝体-内质网-高尔基体，小窝体-内涵体-内质网-高尔基体；P06753-2、P09525、P07148、H0YMD0、H7C469、G3V1A4、P30086、B7Z8M7、P40296、P23141-3、P02766、O95633-2等蛋白调控了寡肽转运体介导的口服聚合物胶束在小肠上皮细胞内的转运。本项目的研究结果既对科学设计和构建肠上皮细胞转运体介导的口服纳米载体具有指导意义，又对阐明其胞内转运的分子调控机制提供了可行的科学方法。因此，本研究既具有非常重要的科学意义，又对实践具有指导作用。