仿生型纳米晶的设计及高效跨胞转运机制的研究

The intestinal epithelium remains a great barrier for oral drug delivery using nanocarriers. The transport of nanocarriers in epithelial cells faces a challenge of "easy entry and hard across", and their intracellular fate is still unclear, therefore significantly restricting the rational design of nanocarrier system for efficient oral drug delivery. This project is proposed based on the mechanism of vesicle transport (i.e. the transport vesicle in cells can specifically recognize, assemble and directionally transport endogenous materials with specific signs) and our previous findings, aiming to design a bio-inspired nanocrystals. This nanocrystals have a very small particle size (below 100 nm), and a functional signal sequence which is bonded to “nanocage” stabilizer via covalent conjugation. This bio-inspired nanocrystals can be significantly endocytosed by intestinal cells, then transport across epithelial cells via transcytosis hijacking transport vesicles, therefore, the transport of drug across intestinal epithelium can be greatly improved, and improving oral absorption. The deep understandings on the mechanism of drug transport across epithelium will be gained by STED super-resolution microscopy, real-time particle tracking , total internal reflection fluorescence microscopy techniques. etc. The findings of this project will dramatically advance our understanding on detailed dynamic trafficking of nanocarriers in cells, and provide solid principles for the rational design of nanocarrier system for oral drug delivery.

消化道上皮细胞屏障是药物纳米载体吸收最重要的生理屏障。药物纳米载体在上皮细胞内转运时面临“入胞容易跨细胞转运难”的科学问题；此外纳米载体的胞内命运不够清晰也严重制约了药物载体的合理设计。本课题基于上皮细胞囊泡定向转运的特殊生理机制（即囊泡能特异性识别、组装并定向转运具有特定信号的胞内物质），结合前期工作基础，拟设计一种仿生型高载药纳米晶。该纳米晶粒径小（<100nm），表面通过“笼状”结构的稳定剂修饰有特殊的信号序列，能高效入胞；入胞后通过特殊信号序列的介导，利用囊泡的转胞吞作用从上皮细胞基底侧胞吐，实现高效跨细胞转运，达到促吸收的目的。本课题采用超分辨率STED显微镜，粒子追踪，全内反射荧光显微镜等技术对转运机制进行系统深入研究，希望为难吸收药物口服纳米载体的合理设计提供依据。

消化道上皮细胞屏障是药物纳米载体吸收最重要的生理屏障。药物纳米载体在上皮细胞内转运时面临“入胞容易跨细胞转运难”的科学问题；此外，纳米载体在胞内命运不够清晰也严重制约了药物载体的合理设计。.为阐明纳米药物在消化道上皮细胞的转运机制，本项目利用基于胞内囊泡转运机制的仿生学原理，设计制备了沙奎那韦脂质纳米晶。研究表明脂质纳米晶在入胞过程中借助脂质筏介导的内吞作用；入胞后脂质修饰改变了纳米药物与囊泡运输系统的相互作用，主要分布于在内质网和高尔基体，更容易进入转运到细胞膜的分泌途径，实现了高效跨细胞转运，显著提高沙奎那韦的口服生物利用度。.其次，本项目以典型的低溶解性BCSⅡ类瑞格非尼为模型药物，制备了不同粒径的纳米晶，并考察了纳米晶粒径对其口服吸收的影响。体外溶解实验显示随着粒径的减小，药物的溶解速度和口服吸收随着粒径的减小而急剧增加，粒径最小的纳米晶可以提高口服生物利用度14倍。此外，本课题设计了一种能搭乘乳糜微粒转运通路的药物自组装载体，该载体携载药物从小肠绒毛AP侧经内质网和高尔基体转运至BL侧，并经淋巴系统进入血液循环，有效提高了瑞戈非尼的口服吸收。.构建了基于FcBP跨胞信号修饰的新型纳米载体，以Caco-2细胞为模型研究了跨胞转运效率和路径。研究结果表明，FcBP信号功能性修饰使得细胞摄取量提高了3.7倍； FMSNs可能通过FcRn介导的胞吞作用进入细胞，入胞后进入早期内涵体，不进入溶酶体。FMSNs在胞内的转运途径与IgG类似，提示FMSNs部分模拟了IgG功能，能够以完整颗粒形式跨胞转运，跨膜转运量具有时间依赖性，在2 h时约有1.1%纳米粒跨过Caco-2细胞单层膜，到6 h时达到4.2%，约为MSNs的16倍。.本项目围绕着解决难吸收药物的口服生物利用度问题，从仿生角度出发，设计了高效的药物递送系统，着重探究其高效定向跨胞转运机制。该项目的实施为理性化设计药物递送系统用于提高口服生物利用度提供了依据。