Abstract：
A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.
摘要：
生物治疗发展的主要障碍是最大化细胞渗透。理想情况下,分析将允许在药物开发过程的早期优化感兴趣的细胞类型中的细胞渗透。然而,存在很少的测定法来比较细胞穿透不同细胞类型而不依赖于药物功能。在这项工作中,我们在7种哺乳动物细胞系以及原代细胞中应用了氯烷烃渗透测定（CAPA）。使用仔细的对照以确保数据可以跨细胞系进行比较。我们比较了几种肽和药物样寡核苷酸的核渗透，发现细胞系之间存在显着差异。为了解释这些差异，我们量化了这些细胞系内吞途径的相对活性，并将它们与渗透数据相关联。基于这些结果,我们在具有有效通透性的细胞系中击倒了网格蛋白，并观察到肽的渗透减少，而不是寡核苷酸。最后,我们使用小分子内体逃逸增强剂，并观察到一些寡核苷酸的细胞渗透增强，但只在一些测试的细胞系中。CAPA数据提供了不同细胞系之间有价值的比较点，包括原代细胞，用于评估各种类型的肽和寡核苷酸的细胞渗透。
