Sébastien Deshayes

Cell-penetrating peptides: tools for intracellular delivery of therapeutics

Abstract.The main problem of therapeutic efficiency lies in the crossing of cellular membranes. Therefore, significant effort is being made to develop agents which can cross these barriers and deliver therapeutic agents into cellular compartments. In recent years, a large amount of data on the use of peptides as delivery agents has accumulated. Several groups have published the first positive results using peptides for the delivery of therapeutic agents in relevant animal models. These peptides, called cell-penetrating peptides (CPPs), are short peptides (fewer than 30 residues) with a net positive charge and acting in a receptor- and energy-independent manner. Here, we give an extensive review of peptide-mediated delivery systems and discuss their applications, with particular focus on the mechanisms leading to cellular internalization.

Cell-penetrating peptides: from molecular mechanisms to therapeutics

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell‐penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG‐ and Pep‐based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure—function relationship of non‐covalent MPG and Pep‐1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

Direct Translocation as Major Cellular Uptake for CADY Self-Assembling Peptide-Based Nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.

Peptide-Based Nanoparticle for Ex Vivo and In Vivo Dug Delivery

One of the major challenges for new therapeutics molecules to enter the clinic remains improving their bioavailability and cellular uptake. Therefore, delivery has become a key stone in therapeutic development and several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs) or protein transduction domain (PTD). PTDs or CPPs were discovered twenty years ago, based on the potency of several proteins to enter cells and nowadays, numerous peptide carriers have been described and successfully applied for ex vivo and in vivo delivery of varying therapeutic molecules. Two CPP-strategies have been reported; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization and the second is based on the formation of stable complexes with drugs depending on their chemical nature. Peptide-Based-Nanoparticle Devices (PBND), correspond to short amphipathic peptides able to form stable nanoparticles with proteins and/or nucleic acids. Three PBND-families, PEP, MPG and CADY have been described, these carriers mainly enter cells independently of the endosomal pathway and efficiently deliver cargoes in a large variety of challenging cell lines as well as in animal models. This review will focus on the structure/function relationship of the PBND: CADY, PEP and MPG, in the general context of drug delivery. It will also highlight the requirement of primary or secondary amphipathic carriers for in vitro and in vivo delivery of therapeutic molecules and provide an update of their pre-clinical evaluation.

Prediction of peptide structure: how far are we?

Rational design of peptides is a challenge, which would benefit from a better knowledge of the rules of sequence–structure–function relationships. Peptide structures can be approached by spectroscopy and NMR techniques but data from these approaches too frequently diverge. Structures can also be calculated in silico from primary sequence information using three algorithms: Pepstr, Robetta, and PepLook. The most recent algorithm, PepLook introduces indexes for evaluating structural polymorphism and stability. For peptides with converging experimental data, calculated structures from PepLook and, to a lesser extent from Pepstr, are close to NMR models. The PepLook index for polymorphism is low and the index for stability points out possible binding sites. For peptides with divergent experimental data, calculated and NMR structures can be similar or, can be different. These differences are apparently due to polymorphism and to different conditions of structure assays and calculations. The PepLook index for polymorphism maps the fragments encoding disorder. This should provide new means for the rational design of peptides. Proteins 2006.

Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery.

Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.

Structural polymorphism of non-covalent peptide-based delivery systems: highway to cellular uptake.

During the last two decades, delivery has become a major challenge for the development of new therapeutic molecules for the clinic. Although, several strategies either viral or non viral have been proposed to favor cellular uptake and targeting of therapeutics, only few of them have reach preclinical evaluation. Amongst them, cell-penetrating peptide (CPP) constitutes one of the most promising strategy and has applied for systemic in vivo delivery of a variety of therapeutic molecules. Two CPP-strategies have been described; using peptide carriers either covalently-linked to the cargo or forming non-covalent stable complexes with cargo. Peptide-based nanoparticle delivery system corresponds to small amphipathic peptides able to form stable nanoparticles with either proteins/peptides or nucleic acids and to enter the cell independently of the endosomal pathway. Three families of peptide-based nanoparticle systems; MPG, PEP and CADY have been successfully used for the delivery of various biologically active cargoes both ex vivo and in vivo in several animal models. This review will focus on the mechanism of the peptide-based nanoparticles; PEP, MPG and CADY in a structural and biophysical context. It will also highlight the major parameters associated to particle formation/stabilization and the impact of the carrier structural polymorphism in triggering cellular uptake.

Interactions of amphipathic CPPs with model membranes

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.

Structural polymorphism of two CPP: an important parameter of activity.

Despite numerous investigations, the important structural features of Cell Penetrating Peptides (CPPs) remain unclear as demonstrated by the difficulties encountered in designing new molecules. In this study, we focused our interest on Penetratin and Transportan and several of their variants. Penetratin W48F and Penetratin W48F/W56F exhibit a reduced and a complete lack of cellular uptake, respectively; TP07 and TP10 present a similar cellular uptake as Transportan and TP08, TP13 and TP15 display no or weak internalization capacity. We applied the algorithmic method named PepLook to analyze the peptide polymorphism. The study reveals common conformational characteristics for the CPPs and their permeable variants: they all are polymorphic. Negative, non permeable, mutants share the opposite feature since they are monomorphic. Finally, we support the hypothesis that structural polymorphism may be crucial since it provides peptides with the possibility of adapting their conformation to medium hydrophobicity and or to partner diversity.