Novel cell penetrating peptides with multiple motifs composed of RGD and its analogs

doi:10.1016/j.bbrc.2013.01.096

Biochemical and Biophysical Research Communications

Volume 432, Issue 2, 8 March 2013, Pages 359-364

https://doi.org/10.1016/j.bbrc.2013.01.096 Get rights and content

Abstract

Cell penetrating peptides (CPPs) have been used to transport macromolecules into cells. Most CPPs have properties such as a strong polycationic charge, amphipathic basic, and hydrophobicity. In this study, we designed the peptides with multiple motifs composed of RGD and its analogs to induce integrin-mediated endocytosis as well as endosomal escape by forming an amphipathic helix in acidic endosomes. These peptides were proved less toxic to animal cells than those without acidic residues. Unexpectedly, peptide conjugated liposomes could penetrate into cells regardless of integrins. The replacement of all aspartic acids by glutamic acids did not prevent the peptide-mediated liposome uptake, and the higher basic and leucine contents enhanced the gene silencing activity of siRNA encapsulated in the liposomes. The peptide is considered to be a new type of CPP which can be used for drug delivery.

Highlights

► Peptides with multiple motifs composed of RGD and its analogs induce liposome uptake into most cells. ► The peptides shows no cytotoxicity at 100 μM. ► The internalized liposome encapsulating siRNA induce the siRNA-mediated gene silencing. ► The peptide with the motifs are considered as a new type of cell penetrating peptide.

Introduction

The plasma membrane prohibits the cellular uptake of exogenous hydrophilic therapeutic molecules, and this makes the cell membrane the first barrier for efficient drug delivery. Short peptide sequences, known as cell penetrating peptides (CPPs), are able to cross the membrane efficiently. CPPs were initially discovered from the short sequences of membrane-interacting proteins called protein transduction domains (PTDs) [1], [2], [3], [4], [5]. These peptide sequences are cationic, hydrophobic, and/or amphipathic in nature [6]. Many CPPs have been designed and predicted in accordance with these properties [7], [8]. Although most amphipathic CPPs are cationic, those with high content of negatively charged residues have also been proved to have cell penetrating activity [9], [10], [11]. However, the cellular uptake of CPPs differs depending on the peptide type. MAP12 and VT5 with the α-helical [9] and the β-sheet structures [10], respectively, penetrated into cells in a non-endocytotic manner, whereas p18 and p28 derived from azurin preferentially entered by endocytotic pathways [11].

RGD peptides containing the arginine–glycine–aspartate motif are able to translocate their cargos into target cells through cell membrane integrin receptors [12]. We initially designed peptides with multiple RGD motifs to have the arginine residues face to one side of the helical-wheel diagram, and then changed some glycine residues to leucine residues in order to increase hydrophobicity (Fig. 1A). Our goal was RGD motif-dependent internalization by cell surface integrins and amphipathic α-helix-dependent endosomal escape in acidic endosomes. Unexpectedly, the peptide internalization was independent of the RGD motif. The peptides could induce liposome internalization, and are considered to be a new type of CPP.

Section snippets

Materials

The peptides were synthesized by the solid-phase method using Fmoc-chemistry. The synthesized peptides were purified by HPLC using a C18 column and analyzed by MALDI-TOF mass spectroscopy (Axima Plus, Shimazu Scientific Instruments, Japan). The Fmoc-amino acids and H-Cys(Trt)-trityl resin were obtained from Novabiochem (Merck KGaA, Germany), and 5-carboxy-X-rhodamine, succinimidyl ester (ROX-SE) for the N-terminal fluorescence labeling of peptide was purchased from Molecular Probes, Inc.

Peptide design and cytotoxicity assay

We designed a peptide (PD1) with multiple RGD motifs to induce RGD/integrin-dependent internalization and to promote endosomal escape by the formation of an amphipathic α-helical structure in acidic endosomes (Fig. 1A). We also designed PD2 with alteration of two glycines to an arginine and a leucine to increase the basic and hydrophobic properties, respectively. We also changed all aspartic acids to glutamic acids of both PD1 and PD2, and named PE1 and PE2, respectively, to remove the RGD

Discussion

Target-specific delivery and endosomal escape of siRNA play important roles in enhancing the therapeutic efficacy of siRNA. We focused on the nature of the RGD motif that is currently used for specific delivery to cancer cells overexpressing integrins, especially αvβ3. Since the amino acid with a carboxylic group has some hydrophobic property in an acidic endosome environment, we initially designed the peptides to localize at different sides of Arg and Asp/Leu on the α-helix wheel diagram (Fig.

Acknowledgments

This research was supported by the Converging Research Center Program of the National Research Foundation of Korea, which is funded by the Ministry of Education, Science and Technology (2012K001395).

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Novel cell penetrating peptides with multiple motifs composed of RGD and its analogs

Abstract

Highlights

Introduction

Section snippets

Materials

Peptide design and cytotoxicity assay

Discussion

Acknowledgments

J. Biol. Chem.

Cell

Cell

J. Biol. Chem.

Drug Discov. Today

Adv. Drug Deliv. Rev.

FEBS Lett.

Cell

Am. J. Pathol.

J. Biol. Chem.

Anal. Biochem.

Cell penetration by transportan

FASEB J.