一种通过内吞作用分析细胞内化机制的通用方法，无非特异性交叉效应,The FASEB Journal

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一种通过内吞作用分析细胞内化机制的通用方法，无非特异性交叉效应
The FASEB Journal ( IF 4.4 ) Pub Date : 2023-01-09 , DOI: 10.1096/fj.202201780r
Mai Itagaki ₁ , Yoshinori Nasu ₂ , Chiaki Sugiyama ₂ , Ikuhiko Nakase ₃ , Noriyasu Kamei ₂

Affiliation

内吞作用是营养吸收和细胞间通讯的重要生物过程；它还可用于加速药物输送载体的细胞内化。阐明未识别的内源性和外源性分子的细胞摄取机制和设计新的有效药物递送系统需要准确、特定的内吞作用分析方法。因此，我们开发了一种通过三种主要内吞途径专门评估细胞内化的方法：网格蛋白和细胞膜穴样内陷介导的内吞作用，以及巨胞饮作用。我们首先揭示了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，我们成功地建立了一种使用小干扰 RNA 来击倒 dynamin-2 和 caveolin-1 的替代方法，在 HeLa 细胞中，这是网格蛋白和细胞膜穴样内陷介导的内吞作用所必需的。第三，我们建立了另一种方法，使用 rottlerin 对 A431 细胞专门分析巨胞饮作用。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，我们建立了多种方法来精确和专门评估新开发的生物制药或药物输送系统的内吞作用。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，我们建立了多种方法来精确和专门评估新开发的生物制药或药物输送系统的内吞作用。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，我们建立了多种方法来精确和专门评估新开发的生物制药或药物输送系统的内吞作用。

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A universal method to analyze cellular internalization mechanisms via endocytosis without non-specific cross-effects

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.

更新日期：2023-01-10

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