Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization

One of the recent hot topics in peptide-related chemical biology research is the potential of cell-penetrating peptides (CPPs). Owing to their ability to deliver exogenous molecules into cells easily and effectively, their flexible design that allows transporters to comprise various chemical structures and functions, and their potential in chemical and cell biology studies and clinical applications, CPPs have been attracting enormous interest among researchers in related fields. Consequently, publications on CPPs have increased significantly. Although there are many types of CPPs with different physicochemical properties and applications, arginine-rich CPPs, which include the human immunodeficiency virus type 1 (HIV-1) TAT peptide and oligoarginines, are among the most extensively employed and studied. Previous studies demonstrated the importance of the guanidino group in arginine, which confers flexibility in transporter design. Therefore, in addition to peptides, various transporters rich in guanidino groups, which do not necessarily share specific chemical and three-dimensional structures, have been developed. Typically, cell-penetrating transporters have 6-12 guanidino groups. Since the pK_a of the guanidino group in arginine is approximately 12.5, these molecules are highly basic and hydrophilic. Our group is interested in why these cationic molecules can penetrate cells. Understanding their mechanism of action should lead to the rational design of intracellular delivery systems that have high efficacy. Additionally, novel cellular uptake mechanisms may be elucidated during the course of these studies. Therefore, our group is trying to understand the basic aspects underlying the ability of these peptides to penetrate cells. Regarding the delivery of biopharmaceuticals including proteins and nucleic acids, achieving efficient and effective delivery to target organs and cells is one of the biggest challenges. Furthermore, when the target sites of these drug molecules are within cells, effective cell penetration becomes another obstacle. Cells are surrounded by a membrane that separates the inside of the cell from its outside. This barrier function is critical for keeping cellular contents inside cells, and without this, cells cannot function. Therefore, understanding the mechanism of action of CPPs is necessary to overcome these obstacles and will allow us not only to improve CPP-mediated delivery but also to create other types of intracellular delivery systems. In this Account, we summarize the current knowledge on the mechanisms of internalization of arginine-rich CPPs, from the viewpoints of both direct cell-membrane penetration (i.e., physicochemical aspects) and endocytic uptake (i.e., physiological aspects), and discuss the implications of this knowledge. We also discussed loosening of lipid packing as a factor to promote direct cell-membrane penetration.