受体的内吞循环对主动靶向制剂胞内转运的影响研究

Among the many factors that may influence the therapeutic effect of active targeting drug delivery system (ATDDS), cellular transport mechanism became a new force in this field. Based on the intracellular transport characteristic of Tfr-Tfn-Fe2+, and combined with our previous study, we suggested that it will make sense to study the impact of receptor recycling on the intracellular transport of ATDDS. In this project, two tasks were proposed to be carried out. The first one is to further study the intracellular transport mechanism of αvβ3-mediated ATDDS. The second is to investigate the intracellular transport mechanism of transferrin receptor-mediated ATDDS, transferrin-modified or 7 pep-modified. The aim of this project was first to illustrate the direct/indirect relationship between the exocytosis of ATDDS and its corresponding receptor. And second, as to the ATTDS retained in the cell, the separation mechanism and the destination of the ligand, receptor and the carrier were supposed to be illuminated, including the separation time, separation site and also related mechanisms. We expected that this study may build the foundation for the development of new ATTDS and the following study on cellular transport mechanism, as well as the efficacy evaluation.

在影响主动靶向制剂药效的诸多因素中，细胞转运机制对药效的影响成为近年来药学工作者的关注热点。受Tfr-Tfn-Fe2+胞内转运机制的启发，同时结合我们的前期研究结果，我们创新性地提出从细胞分子水平出发，考察受体的内吞循环对主动靶向制剂胞内转运的影响。主要研究内容包括：① 进一步完善αvβ3受体介导的纳米粒细胞转运机制的考察；② Tfr介导的两种纳米粒（Tfn修饰和7pep修饰）的细胞转运机制考察。重点阐明：① 主动靶向制剂的外排与受体循环转运的直接/间接相关性；② 未外排而滞留在胞内的制剂，配体、受体、制剂解离的时间——when、位置——where、去向，以及解离的相关机制——how。本项目的研究结果，以及建立的研究方法，将对今后主动靶向制剂的设计优化以及后续的药效学评价提供重要的参考。

在影响主动靶向制剂药效的诸多因素中，细胞转运机制对药效的影响成为近年来药学工作者的关注热点。受Tfr-Tfn-Fe2+胞内转运机制的启发，同时结合我们的前期研究结果，我们创新性地提出从细胞分子水平出发，考察受体的内吞循环对主动靶向制剂胞内转运的影响，首先完善了αvβ3受体介导的cRGDfK-PLGA-NPs的细胞转运机制；其次考察比较了Tfr介导的两种纳米粒（Tfn修饰和7pep修饰）的细胞转运机制。结果表明，从胞外向早期内吞体的转运，主动组的转运速率常数均高于被动组，且7pep 修饰的胶束从胞外进入早期内吞体的转运速率常数要高于Tfn修饰的胶束，说明7pep-PCL胶束入胞速率快于Tfn-PCL胶束。入胞后，与被动组相比，cRGDfK-PLGA-NPs从早期内吞体向内吞循环隔室转运的速率常数，以及从晚期内吞体到溶酶体转运的速率常数均明显小于被动组，可见 cRGDfK的修饰会引起纳米粒的溶酶体逃逸，存在明显的细胞外排过程，且与早期内吞体相关，cRGDfK修饰的纳米粒会在αvβ3受体的介导下通过快循环途径出胞。在Tfr介导的纳米粒的胞内转运，7pep修饰的胶束从早期内吞体向内吞循环隔室的转运速率常数和从晚期内吞体向溶酶体转运的速率常数可以看出，胶束在7pep 的修饰下，亦可以逃离溶酶体的降解，这提示我们7pep的修饰可能存在与cRGDfK修饰同样的受体介导的纳米粒的外排行为。 而Tfn修饰的胶束则表现出与被动组相似的通过溶酶体降解路径，即在内吞循环隔室中聚集后，会进一步转运进入溶酶体中进行降解。本项目的研究结果，以及建立的研究方法，将对今后主动靶向制剂的设计优化以及后续的药效学评价提供重要的参考。