Oligoarginine vectors for intracellular delivery: Role of arginine side-chain orientation in chain length-dependent destabilization of lipid membranes

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Abstract

Arginine-rich peptides receive increased attention due to their capacity to cross different types of membranes and to transport cargo molecules inside cells. Even though peptide-induced destabilization has been investigated extensively, little is known about the peptide side-chain and backbone orientation with respect to the bilayer that may contribute to a molecular understanding of the peptide-induced membrane perturbations.

The main objective of this work is to provide a detailed description of the orientation of arginine peptides in the lipid bilayer of PC and negatively charged PG liposomes using ATR-IR spectroscopy and molecular modeling, and to relate these orientational preferences to lipid bilayer destabilization.

Molecular modeling showed that above the transition temperature arginine side-chains are preferentially solvent-directed at the PC/water interface whereas several arginine side-chains are pointing towards the PG hydrophobic core. IR dichroic spectra confirmed the orientation of the arginine side chains perpendicular to the lipid–water interface. IR spectra shows an randomly distributed backbone that seems essential to optimize interactions with the lipid membrane. The observed increase of permeation to a fluorescent dye is related to the peptide induced-formation of gauche bonds in the acyl chains. In the absence of hydrophobic residues, insertion of side-chains that favors phosphate/guanidium interaction is another mechanism of membrane permeabilization that has not been further analyzed so far.

Highlights

► We investigate the side-chain and backbone orientation of poly-arginine in phospholipid bilayers. ► Above the transition temperature, Arg7 and Arg4, but not Arg1, are found to destabilize PG bilayers. ► Certain side-chains are found to be directed towards the hydrophobic core in PG but not in PC bilayers. ► The interaction results in an increase in gauche conformations of the acyl chains with a general increase of bilayer disorder.

Introduction

Cell penetrating peptides (CPPs) display the ability to cross cell membranes and transport cargo molecules inside cells. One of the most representative members of this family of peptides is a short, arginine-rich peptide segment derived from the human immunodeficiency virus (HIV)-1Tat protein. Cellular uptake mechanisms remain nevertheless controversial. For instance, it has been suggested that the TAT peptide translocates with its cargo into eukaryotic cells through a physical mechanism that is not receptor-mediated, without implicating the endosomal pathway (Henriques et al., 2005). This suggests that translocation would require a rearrangement of the lipid bilayer organization and/or packing. In contrast, other studies propose a raft-dependent endocytic pathway, involving macropinocytosis (Wadia et al., 2004). These membrane-permeable peptides share little similarity in their primary and secondary structures except for a high concentration of arginine residues in their sequences. In addition, various arginine-rich oligopeptides display very similar properties in terms of translocation and delivery efficiency, suggesting an obvious correlation between translocation and arginine content (Futaki, 2006). The translocation may be a consequence of the interaction between the hydrophilic moiety of phospholipids and the side-chains of arginine residues as suggested by the arginine-rich peptides’ length-dependent internalization and the absence of internalization of peptides with three arginine residues or less (Tung and Weissleder, 2003).

Whereas part of CPPs uptake might involve specific receptors, internalization is observed even in their absence (Richard et al., 2005). Furthermore, these peptides can enter giant unilamellar vesicles made exclusively of lipids (Binder and Lindblom, 2003). All together these data strongly suggest that a specific interaction between the hydrophilic moiety of phospholipids and the side-chains of arginine residues is an important step for peptide internalization.

The interaction between the CPPs and the cell membrane is the first step involved in the uptake mechanism. Even though peptide-induced membrane perturbations has been investigated extensively, little information is available about the orientation of the peptide side-chains and backbone with respect to the bilayer that may open the way to a molecular understanding of the peptide-induced membrane perturbations. The fact that the hydrophobic residues present in the sequence of these penetrating peptides favored penetration is possibly a direct consequence of an increased hydrophobicity. The challenge of this work is to deal with highly hydrophilic peptide and to understand how it could nevertheless destabilize a lipid bilayer. Therefore, the main objective of this work is to provide a description of the backbone and side-chain orientation of Arginine oligopeptides (Arg4, Arg7) in the lipid bilayer, in comparison with the isolated amino acid (arg+), using ATR-IR spectroscopy and molecular dynamics simulation and to relate orientation preferences of the peptide with the lipid bilayer packing and organization. In this sense we use zwitterionic PC and negatively charged PG because we want to reveal the mechanism of binding which always precedes a possible translocation, focusing on the influence that peptide binding has on lipid membrane properties. The phosphate moiety was identified in previous studies as a prominent factor in arginine–lipid interaction and therefore DMPG appeared as a valuable biophysical model (Sakai et al., 2005, Tang et al., 2007).

Section snippets

Materials

DMPC, DMPG, DOPG were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used as received. Peptides and amino-acids: Arg7, Arg4 were from AnyGen Co. Ltd., and l-arginine (arg+) was obtained from Sigma–Aldrich (Saint Louis, MO).

Attenuated total reflection Fourier transform IR spectroscopy (ATR-FTIR)

The internal reflection element was a 52 mm × 20 mm × 2 mm trapezoidal germanium ATR plate with an aperture angle of 45° yielding 25 internal reflections. Infra-red spectra were recorded on an IFS55 FTIR spectrophotometer (Bruker, Ettlingen, Germany) purged with N2. Fifteen microliters of the liposomes sample were deposited under a stream of nitrogen on one side of the germanium. While evaporating, capillary forces flattened the membranes which spontaneously formed oriented multilayer arrangements.

ATR-IR

The transition from gel to liquid crystalline phase causes conformational disorders in the acyl chains (i.e., gauche rotamers) and rearrangements in the interfacial and polar head group regions, leading to major changes in the infrared spectrum. IR bands in the 3000–2800 cm−1 region arise predominantly from the symmetrical and asymmetrical CH2 and CH3 stretching vibrations and are quite sensitive to temperature-induced changes. The frequency, the intensity, and the width of these bands

Discussion

Comparison of different backbones showed that a main requirement for cellular uptake is an unordered backbone; this parameter seems essential to optimize interactions with the lipid membrane. The absorption band shift from about 1651–1655 cm−1 to about 1644–1646 cm−1, regardless of the chain length of the peptide, is indicative of a backbone adopting a random structure and confirms these assumptions. The fact that no leakage is observed with free arginine indicates that even though electrostatic

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