Colloids and Surfaces B: Biointerfaces 106 (2013) 240–247 Contents lists available at SciVerse ScienceDirect Colloids and Surfaces B: Biointerfaces jou rnal h om epa g e: www.elsevier.com/locate/colsurfb The activity of LE10 peptide on biological membranes using molecular dynamics, in vitro and in vivo studies Egipto Antunes a , Nuno G. Azoia a , Teresa Matamá a,b , Andreia C. Gomes b , Artur Cavaco-Paulo a,∗ a Biological Engineering Department, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal b Molecular and Environmental Biology Centre (CBMA), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal a r t i c l e i n f o Article history: Received 6 September 2012 Received in revised form 16 January 2013 Accepted 17 January 2013 Available online 4 February 2013 Keywords: Antimicrobial peptides Cell-penetrating peptides Liposome disruption Membrane active peptides Molecular dynamics a b s t r a c t Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are generally defined as small cationic peptides with the ability to interact with lipidic membranes, in a process driven by electro- static and hydrophobic processes. The interaction with CPPs is known to lead to its translocation across the membrane, while with AMPs lead to membrane damage. Here we present one synthetic anionic peptide, LE10 (LELELELELELELELELELE), which strongly interacts with model membranes, showing properties of CPPs (translocation through lipidic membranes on a mechanism usually described for cationic CPPs) and AMPs (membrane disruption) in molecular dynamic studies, experimental studies with liposomes and mammalian cells in vitro. Based on the LE10 properties here demonstrated, small modifications in its structure could make it a very promising tool for drug delivery. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two groups of short peptides with the ability to interact with cell membranes with different outcomes. CPPs are molecules that rapidly internalize across cell mem- branes and can transport macromolecules into living cells [1]. These molecules are, generally, short peptides (up to 30 amino acids) with positive net charge (rich in lysines and/or arginines) [2]. The mech- anism of translocation of the membrane by CPPs remains unclear, and three main entry mechanisms have been proposed in the litera- ture, namely direct translocation [3], different forms of endocytosis, such as macropinocytosis [4], clathrin-mediated endocytosis [5] and caveolae/lipid raft-mediated endocytosis [6], and penetration through a transitory structure, like micelles [7] and pores [8]. AMPs, in turn, are antibiotic molecules, having the ability to kill a broad spectrum of microorganisms and cells, like bacteria, para- sites, tumor cells, fungi and viruses [9,10]. In most cases, AMPs are cationic, and interact with the target membrane surface, in a pro- cess involving interactions between the charged residues of the peptides and anionic components of the cell membrane surface [10], killing the target by membrane disruption or permeabiliza- tion, or translocating membrane and killing the target by other mechanisms. Some of these mechanisms are inhibition of DNA ∗ Corresponding author. Tel.: +351 253604400; fax: +351 253604429. E-mail address: artur@deb.uminho.pt (A. Cavaco-Paulo). or RNA synthesis, blocking of chaperones, inhibition of ribosomal activity, and induction of reactive oxygen species or disruption of mitochondrial cell membrane [11]. Unlike CPPs, the existence of negatively charged AMPs is well established, and this type of AMPs has been known since 1980 [12]. Thus, according to their charge, AMPs can be classified as cationic AMPs (CAMPs), generally with a net charge in the range of +4 to +6 due to presence of lysine and arginine, or as anionic AMPs (AAMPs), normally with a net charge in the range of -1 to -7, due to glutamic and aspartic acid residues. Although AAMPs have been described in a wide range of orga- nisms, from vertebrates to plants [12], the number of studies with these peptides is very low, compared with CAMPs, and their method of action is still unclear. On the other hand many models of CAMPs membrane activity have been proposed. The most common are the barrel stave pore model [13], the toroidal pore model [14], the carpet model [15] and the detergent model [16]. To our knowledge there is only one anionic CPP (ACPP) described, the “SAP(E)” (VELPPPVELPPPVELPPP). The SAP(E) is a synthetic peptide and results of one investigation about the role of peptides net charge in its cell-penetrating properties [17]. Starting from a well-known CPP, SAP (VRLPPPVRLPPPVRLPPP), the authors changed the positive arginine by glutamic acids. The results clearly show that the positive net charge is not a fundamental feature to the SAP activity, since the SAP(E) showed similar uptake efficiency and mechanism of action. It is expected that this fact may also be true for other CPPs. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.050