Itagaki Mai, Nasu Yoshinori, Sugiyama Chiaki, Nakase Ikuhiko, Kamei Noriyasu
Laboratory of Drug Delivery Systems, Graduate School of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan.
Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan.
FASEB J. 2023 Feb;37(2):e22764. doi: 10.1096/fj.202201780R.
Endocytosis is an essential biological process for nutrient absorption and intercellular communication; it can also be used to accelerate the cellular internalization of drug delivery carriers. Clarifying the cellular uptake mechanisms of unidentified endogenous and exogenous molecules and designing new effective drug delivery systems require an accurate, specific endocytosis analysis methodology. Therefore, we developed a method to specifically evaluate cellular internalization via three main endocytic pathways: clathrin- and caveolae-mediated endocytosis, and macropinocytosis. We first revealed that most known endocytosis inhibitors had no specific inhibitory effect or were cytotoxic. Second, we successfully established an alternative method using small interfering RNA to knock down dynamin-2 and caveolin-1, which are necessary for clathrin- and caveolae-mediated endocytosis, in HeLa cells. Third, we established another method to specifically analyze macropinocytosis using rottlerin on A431 cells. Finally, we validated the proposed methods by testing the cellular internalization of a biological molecule (insulin) and carriers (nanoparticles and cell-penetrating peptides). Through this study, we established versatile methods to precisely and specifically evaluate endocytosis of newly developed biopharmaceuticals or drug delivery systems.
内吞作用是营养物质吸收和细胞间通讯的重要生物学过程；它还可用于加速药物递送载体的细胞内化。阐明未知内源性和外源性分子的细胞摄取机制以及设计新的有效药物递送系统需要一种准确、特异的内吞作用分析方法。因此，我们开发了一种方法，通过三种主要的内吞途径特异性评估细胞内化：网格蛋白介导的内吞作用、小窝蛋白介导的内吞作用和巨胞饮作用。我们首先发现，大多数已知的内吞作用抑制剂没有特异性抑制作用或具有细胞毒性。其次，我们成功建立了一种替代方法，利用小干扰RNA敲低HeLa细胞中网格蛋白介导的内吞作用和小窝蛋白介导的内吞作用所必需的发动蛋白2和小窝蛋白1。第三，我们建立了另一种方法，使用rottlerin在A431细胞上特异性分析巨胞饮作用。最后，我们通过测试生物分子（胰岛素）和载体（纳米颗粒和细胞穿透肽）的细胞内化来验证所提出的方法。通过这项研究，我们建立了通用的方法来精确、特异性地评估新开发的生物药物或药物递送系统的内吞作用。
