Salim Heba, Pei Dehua
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2022;2371:301-316. doi: 10.1007/978-1-0716-1689-5_16.
Intracellular biologics such as cyclic peptides are an emerging class of macromolecular drugs that are either intrinsically cell permeable or can be effectively delivered into the cell interior to modulate the activity of previously intractable drug targets. They generally enter the mammalian cell by endocytosis mechanisms and are initially localized inside the endosomes. They subsequently escape from the endosomes (and/or lysosomes) into the cytosol with varying efficiencies. In this chapter, we provide the detailed protocol for a flow cytometry-based assay method to quantitate the overall cellular uptake, endosomal escape, and cytosolic entry efficiencies of biomolecules (e.g., linear and cyclic peptides, proteins, and nucleic acids), by using cell-penetrating peptides as an example. The scope of applicability, strengths, and weaknesses of this assay are also discussed.
细胞内生物制剂，如环肽，是一类新兴的大分子药物，它们要么本质上具有细胞渗透性，要么可以有效地递送至细胞内部，以调节先前难以处理的药物靶点的活性。它们通常通过内吞作用机制进入哺乳动物细胞，并最初定位于内体内部。随后，它们以不同的效率从内体（和/或溶酶体）逃逸到细胞质中。在本章中，我们以细胞穿透肽为例，提供了一种基于流式细胞术的检测方法的详细方案，用于定量生物分子（如线性和环肽、蛋白质和核酸）的整体细胞摄取、内体逃逸和细胞质进入效率。还讨论了该检测方法的适用范围、优点和缺点。
