Macrocyclic Cell Penetrating Peptides: A Study of Structure- Penetration Properties Hassan Traboulsi, † Heidi Larkin, † Marc-Andre ́ Bonin, † Leonid Volkov, ‡ Christine L. Lavoie, † and E ́ ric Marsault* ,† † Institut de Pharmacologie de Sherbrooke, Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences and ‡ Biophotonics Core Facility, Centre de Recherche du Centre Hospitalier, Universite ́ de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada * S Supporting Information ABSTRACT: Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internal- ization. Particularly, backbone rigidification, including macro- cyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure-uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells. ■ INTRODUCTION Cell-penetrating peptides (CPPs), originally known as protein transduction domains, are peptides consisting of fewer than 35 amino acids capable of penetrating cells despite their peptidic character. Initially identified as specific transduction domains embedded in protein sequences, they represent a natural mechanism of cellular targeting and entry. 1-4 Since their discovery, the cell penetrating properties of CPPs have been harnessed to internalize otherwise impermeable cargos into cells, including small molecules, macromolecules (e.g., proteins, nucleic acids), and nanoparticles. 5-11 The majority of CPPs contain a high relative abundance of positively charged amino acids such as lysine (Lys) or arginine (Arg), or display alternating patterns of polar/charged amino acids and nonpolar, hydro- phobic amino acids. 9,12,13 Arginine-rich CPPs have been used successfully to deliver a broad diversity of biologically active macromolecules intra- cellularly, although their mechanisms of cellular entry are still under investigation. 9,12,14-17 In the process, the guanidinium cationic groups of the Arg residues are crucial for efficient cell penetration, a role attributed to their capacity to interact with the various anion types localized on the plasma membrane such as the polar heads of phospholipids or the sulfate groups of glycosaminoglycans. 13,18,19 Polyarginines are readily synthesized and represent the structurally simplest CPPs. They have been studied extensively for their ability to penetrate through cell membranes. It was found that cellular uptake can be achieved with oligoarginines composed of 5-15 residues. 14,20 In particular, nona-arginine (R 9 ) was shown to possess improved cell penetration efficiency compared to TAT peptides. 9,20 Thus, several studies have utilized R 8 and R 9 as reference tools to import into cells a variety of biological molecules including siRNA, anticancer drugs, small molecules, proteins, peptides, and oligonucleotides. 10,11 The mechanisms by which CPPs translocate across cell membranes remain a subject of investigation. Certain CPPs, when attached to small cargos, translocate passively across the plasma membrane of cells. 21 However, an increasing number of studies have shown that cellular uptake of cationic CPPs, when conjugated to macromolecules or used at low concentrations, is temperature-sensitive, suggesting the involvement of energy- dependent processes. 17,22-25 To better delineate the mecha- nisms at play, Matsushita et al. recently applied siRNA library screening to identify potential partners involved in the cellular entry of polyarginine peptides. This led to the identification of Received: July 3, 2014 Revised: February 5, 2015 Published: February 5, 2015 Article pubs.acs.org/bc © 2015 American Chemical Society 405 DOI: 10.1021/acs.bioconjchem.5b00023 Bioconjugate Chem. 2015, 26, 405-411