基于共培养细胞模型的纳米载药系统细胞转运实时示踪分析

Nano drug delivery system has been one of the most advanced administrations for clinical therapy. Nano delivery system exhibits numerous advantages due to alter dynamics processes of drug in vivo. However there are a few questions in complex environment in vivo, for example, whether nano drug delivery system can pass through many barriers consisting of different epithelium cells and connective tissues before they are taken up by the target cells, and whether they are stable in the process of crossing these barriers. In addition, it that the research about the interaction between nano drug delivery system and cell becomes urgent need to solve a scientific problem for pharmaceutical analysis. So, the further study about the mechanisms of adsorption, uptake and trans-membrane of nano system in cell are very meaningful to interpret the behavior of nano drug delivery system in vivo. This study will be based on the design concept of “a co-culture cell model and a double fluorescence labeling technique” and focus on the key link of “the transport processes of the whole nano drug delivery system in a co-culture cell model”. The aim is to develop a non-invasive, real-time and sensitive analysis method using the fluorescence molecular imaging technology, and this method can be used for continuously and dynamically monitoring the behaviors of nano drug delivery system in living cell. In this study, the interaction between nano drug delivery system and cell surface will be analyzed, the pathway of endocytosis and efflux of nano system in cell and the characteristics of the trans-epithelial cell transport will be also investigated. Moreover, the dynamic processes of nano system in cell will be visualized. A new idea and way will be provided for study the behavior of nano drug delivery system in living cell.

纳米载药系统是当今临床治疗最具先进性的给药形式之一，通过改变药物的体内过程而表现出诸多优越性。但复杂的体内环境，使纳米载药系统需要跨越由不同细胞构成的多种生物屏障，才能将药物转运到靶部位；而纳米系统与细胞屏障发生相互作用后的稳定性，也使其细胞内过程分析成为药物分析领域的一个研究瓶颈，因此，清楚解析纳米系统的细胞转运机理对于阐释其体内行为具有重要意义。本研究基于“双细胞+双示踪”的设计理念，着眼于“纳米载药系统整体的多细胞转运行为”这一关键环节，构建双标记纳米载药系统和双细胞转运模型，建立一种非侵入式、实时、灵敏的细胞水平的荧光示踪分析方法。综合分析纳米载药系统与细胞质膜的相互作用、被细胞内吞/外排的途径、经共培养细胞模型转运的特点和动力学过程，以期阐明纳米系统在细胞内的行为和跨膜转运的机理，为纳米载药系统在细胞水平的过程研究提供新思路和新方法。

纳米载药系统是当今临床治疗最具先进性的给药形式之一，通过改变药物的体内过程而表现出诸多优势。但复杂的体内环境，使纳米载药系统需要跨越由不同细胞构成的多种生物屏障，才能将药物转运到靶部位。因此，清楚解析纳米载药系统的细胞转运行为和机理是探明其体内行移过程的首要科学问题之一。. 为探明这一科学问题，项目规划了3个研究专题：示踪生物相容性材料的制备和双示踪纳米载药系统的构建及特性表征；双示踪纳米载药系统经细胞黏附、摄取动力学行为分析和机理研究；Caco-2细胞单层模型、Caco-2/EA.hy926共培养细胞模型的构建和纳米载药系统转运行为分析和差异性研究。获得如下研究结果：（1）以罗丹明B与聚乳酸-羟基乙酸共聚物化学合成了荧光生物材料，为解析纳米粒细胞行为提供了表征方法；（2）构建双荧光纳米载药系统、考察了环境因素对其光学特性的影响，实现了包载药物和载体材料共同示踪纳米粒的目标；（3）构建了Caco-2/EA.hy926共培养细胞模型，用于研究纳米载药系统从小肠吸收、穿过血管、进入血液循环的多细胞跨膜转运过程；（4）综合荧光显微成像技术和荧光定量分析方法表征了纳米载药系统的细胞动力学行为，不仅可同时呈现双荧光纳米粒包载物的绿色荧光、载体材料的红色荧光和纳米粒的重叠黄色荧光；还可以准确、快速测定包载物和载体材料的含量；（5）清楚解析了纳米粒在多细胞跨膜转运过程中药物释放、纳米粒裂解等行为，并发现EA.hy926细胞对大分子量的载体材料有转运阻滞作用。. 项目实施所设计合成的示踪生物材料材料，工艺简单可行，专利已获授权，可开发为一种新的功能性高分子材料；构建的双荧光纳米载药系统可实现以基本结构单元形象、真实示踪纳米粒；建立的Caco-2/EA.hy926共培养细胞模型，为药物的吸收特征研究提供了一种更接近体内过程的体外研究模型；基于“双标记纳米载药系统和双细胞转运模型”的项目研究，阐释了纳米载药系统的多细胞跨膜转运过程，为纳米系统的细胞行为研究提供了新的思路和方法。

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