The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

Yan Ma,Cheng Gong,Yilong Ma,Yu-Hui Zhang

doi:10.1142/s1793545814500199

Abstract

D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro and in vivo application. Nevertheless, their internalization pathway is obscure and conflicting results have been obtained concerning their intracellular distribution. In this study, we demonstrate that octa-D-arginine (r8) undergoes diffuse localization throughout the cytoplasm and nucleus even at low concentrations and that r8(r: D-arginine) enters the cells via direct membrane translocation, unlike R8(R: L-arginine), of which endocytosis is the major internalization pathway. The observation that R8and r8enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry affects the uptake mechanism of oligoarginines.

Highlights

A number of therapeutic molecules available for intracellular targets is severely limited by the general requirement that they must breach cell membranes
Among the existing Cell-penetrating peptides (CPPs), homogeneous oligoarginine peptides (L or D) are of particular interest,[4,5] they are the most widely studied and frequently employed CPPs in both in vitro and in vivo applications because they are accessibly synthesized and oligomers as short as 7 $ 12 amino acid residues are of high cellular uptake e±ciency.[6,7,8,9,10,11,12,13,14,15]
We demonstrate that r8 enters the cells by direct membrane translocation rather than endocytosis and becomes di®usely localized throughout the cytosol and nucleoli, rather than remaining trapped in endosomal vesicles

Summary

Introduction

A number of therapeutic molecules available for intracellular targets is severely limited by the general requirement that they must breach cell membranes. Compared with their L-counterparts, D-arginine oligomers have been reported to possess a higher transduction and greater protease resistance[4,20] and are used more often in in vivo applications.[10] But in contrast, there are much fewer studies on of D-arginine oligomers To this point, most of the studies are focused on the cellular uptake e±ciency of Darginine oligomers.[4,20] the high cellular uptake e±ciency of CPPs does not always relate to high functional e®ectiveness of their cargos once inside the cell.[21,22] The intracellular distribution and internalization pathway employed in°uence the destination and biological e±cacy of intracellularly delivered therapeutic agents. Our results suggest that the backbone stereochemistry a®ects the cellular uptake mechanism of oligoarginine peptides

Synthesis of peptides

Confocal microscopy

Incubation with inhibitors of endocytosis

Results