Current Pharmaceutical Design - Volume 19, Issue 16, 2013

Since the initial evidence that antennapedia homeobox [1] can cross cell membranes and internalize into cells, numerous peptides with similar translocation properties have been described. These peptides are referred to as cell-penetrating peptides (CPPs) or protein- transduction domains (PTDs). Reviews on reported CPP sequences have been recently published [2,3], together with reviews on their mechanisms of internalization [2,4-6]. In this review, we will focus on natural homeoproteins and homeoprotein-derived peptides and describe results that have been obtained among different laboratories to unravel the different pathways by which these molecules reach the cell cytosol and nucleus or transfer from one cell to another. Using homeoproteins as a paradigm, we will also summarize recent evidences of the physiological functions of endogenous protein translocation.

Intracellular delivery using cell-penetrating peptides (CPPs) has received considerable attention as a promising method for introducing exogenous molecules into cells. The mechanisms that enable efficient internalization of CPPs together with cargo molecules are also of interest. In this review, we describe our current views of membrane translocation of CPPs, especially those rich in arginine, as these peptides represent one of the most frequently employed classes of CPPs.

Although siRNA consist in very promising therapeutics, their clinical development is limited by several biological barriers including low cellular permeability, poor stability and lack of tissue specificity. Therefore the Achilles' heel for siRNA-based therapy is directly related to the lack of efficient system to promote their delivery. During the last two decades, cell-penetrating peptides (CPPs) have been widely developed to enhance the cellular delivery of therapeutics. In this context we have elaborated a new strategy based on selfassembling peptide-based nanoparticles. The CADY peptide is a 20-residue secondary amphipathic peptide which is able to spontaneously self associate with siRNA with a strong affinity, by combining both electrostatic and hydrophobic interactions, to form stable nanoparticles. Investigations of both physico-chemical properties and cellular siRNA delivery revealed that the CADY/siRNA complexes were able to enter a wide variety of cell lines by a mechanism independent of any endocytotic pathway. In addition a deeper understanding of the self assembly of CADY molecules around siRNA leads to a "raspberry"-like nanoparticle architecture which provides new perspectives for the CADY/siRNA formulations. Finally the robustness of the biological response infers that peptide-based nanoparticle technology holds a strong promise for therapeutic applications. The present review deals with most of the biophysical characteristics as well as the cellular mechanism and cellular applications of CADY/siRNA nanoparticles.

The insides of cells can be viewed as a treasure trove of targets for therapeutic intervention of diseases or as deposits for contrasting agents. Increasingly the molecules that need to be delivered to the inside of cells for these purposes are macromolecular and membrane impermeable. Cell penetrating peptides (CPPs) have proven abilities to deliver a range of macromolecular cargo into cells thus raising their profile as potential delivery vectors for wide-ranging applications. There is evidence to suggest that CPPs first enter cells through endocytosis and that cytosolic delivery is mediated across endolysosomal membranes. Their capacity to do this, over direct plasma membrane translocation, is likely to depend on the nature and size of the cargo. Cells use a range of endocytic routes to facilitate entry from well characterised pathways regulated by clathrin to more recently discovered and less characterised pathways regulated by clathrin independent mechanisms. These are likely to determine the intracellular fate of cell delivery vectors including those based on cell penetrating peptides. Thus gaining accurate knowledge of their endocytic uptake and traffic is an important characterisation criteria for progress in this field. This review describes the different endocytic pathways that have been identified in mammalian cells and specific reports that have studied the uptake mechanisms and endocytic traffic of cell penetrating peptides and their associated cargo. These cargoes range from short peptides to an increasing library of nanoparticles such as quantum dots, liposomes and polymeric dendrimers. The studies highlight the effectiveness of cell penetrating peptides for delivering these entities into a diverse array of cell types using different endocytic pathways. This is shown using microscopy based colocalisation analysis with the few specific endocytic probes available, and chemical inhibitors of endocytosis that suffer from lack of specificity. Overall, more specific probes, inhibitors and novel technologies are required for accurate characterisation of cellular dynamics of cell penetrating peptide conjugates thus allowing them to reach their full potential as vectors for therapeutics and other payloads.

The increasing knowledge on the genetic basis of disease has allowed the development of promising gene-targeted therapies that can be applied to numerous diseases. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or repair and regulation of specific genes. However, the clinical application of these large and charged molecules remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. Since the first observations, two decades ago, that some protein-derived domains can translocate across biological membranes, a wide group of peptides called cellpenetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. The mechanistic basis of CPP and CPP conjugate cellular uptake remains controversial. However, biophysical studies on the interactions of CPPs with membrane models have contributed to unravel the mechanisms underlying CPP membrane translocation as well as to propose relationships between those mechanisms and CPP efficiency in mediating cargo delivery. In this review, representative examples of CPPs were gathered from the most recent literature in order to emphasize the contributions of chemists, biophysicists and cell biologists towards the rational design of increasingly more efficient delivery systems. In this context, the present review aims at giving an overview of some of the most significant CPP families and their biological applications as nucleic acid delivery systems.

Cell-penetrating peptides (CPPs) are promising tools for intracellular delivery. However, no consensus regarding their internalization mechanism has been reached within the scientific community. Although endocytosis seems to be the preferred internalization mechanism for most CPP-cargo complexes, examples of direct translocation have also been identified. In this review we go through the several ways of studying CPP-mediated cargo delivery in cells and the possible factors affecting the internalization pathways followed by these complexes. In addition, we analyze the CPP-mediated delivery of two relevant cargoes, namely quantum dots and nucleic acids, focusing on the internalization mechanism that they follow.

siRNA-induced RNA Interference (RNAi) responses have great potential to treat human disease; however, siRNAs are highly charged macromolecules with no ability to enter cells and require a delivery agent. Peptide Transduction Domains (PTDs), also called Cell Penetrating Peptides (CPPs), are delivery peptides with the potential to deliver macromolecular peptides, proteins and siRNAs into cells. Here we discuss the multitude of ways that PTDs/CPPs have been utilized to deliver siRNAs from direct conjugates to complex nanoparticles. PTD/CPP-mediated siRNA delivery has come a long way and has great potential to address the siRNA delivery problem.

Duchenne muscular dystrophy is a severe, X-linked muscle wasting disorder caused by the absence of an integral structural protein called dystrophin. This is caused by mutations or deletions in the dystrophin gene which disrupt the reading frame, thereby halting the production of a functional protein. A number of potential therapies have been investigated for the treatment of this disease including utrophin upregulation, ‘stop-codon read through’ aminoglycosides and adeno-associated virus gene replacement as well as stem cell therapy. However, the most promising treatment to date is the use of antisense oligonucleotides which cause exon skipping by binding to a specific mRNA sequence, skipping the desired exon, thereby restoring the reading frame and producing a truncated yet functional protein. The results from recent 2’OMePS and morpholino clinical trials have renewed hope for Duchenne patients; however in vivo studies in a mouse model, mdx, have revealed low systemic distribution and poor delivery of oligonucleotides to affected tissues such as the brain and heart. However a variety of cell penetrating peptides directly conjugated to antisense oligonucleotides have been shown to enhance delivery in Duchenne model systems with improved systemic distribution and greater efficacy compared to ‘naked’ antisense oligonucleotides. These cell penetrating peptides, combined with an optimised dose and dosing regimen, as well as thorough toxicity profile have the potential to be developed into a promising treatment which may be progressed to clinical trial.

Morpholino oligos (Morpholinos) are widely used tools for knocking down gene expression and are currently in a clinical trial for treatment of Duchene muscular dystrophy. A Morpholino analog has been in a clinical trial as a potential anti-bioterrorism agent for inhibiting replication of deadly Marburg viral infection. The cellular uptake of Morpholinos can been greatly increased by conjugation with cell-penetrating peptides (CPP). The use of the CPP-Morpholino conjugates (PPMOs) in vivo has been broadly demonstrated in viral, bacterial, genetic and other diseases. The following aspects of PPMOs will be discussed in this paper including chemistry, stability, antisense specificity, mechanism of cellular uptake, in vivo efficacy, tissue distribution, pharmacokinetics, toxicity and the human clinical trials. PPMOs are powerful research tools for studying gene function in animals and their properties are being improved as potential human therapeutic agents.

Acute myocardial infarction (AMI) is a frequent and disabling disease, which is the first cause of cardiovascular mortality worldwide. Infarct size is a major determinant of myocardial functional recovery and mortality after AMI. Limitation of infarct size thus appears as an appropriate strategy to prevent post-ischemic heart failure and improve survival. Reperfusion is the only treatment recommended to reduce infarct size but despite obvious benefits, it may also have deleterious effects called ischemia-reperfusion (IR) injury including myocyte cell death. Proteins involved in the apoptosis cascade generally interact over large surfaces lacking well-defined pockets. Therefore, inhibitory peptides are optimal biomolecules to target these large protein surfaces, they are often more selective to their target than conventional small organic molecules, and they can be tailored for optimal affinity or desired metabolic property. Since peptides do not cross freely biological membranes, they are generally administered in association with cell penetrating peptides (CPPs) and with homing peptides (HPs) for selective organs or tissues targeting. As a first approach in vivo, we made use of the already known BH4 peptidic inhibitor of the mitochondrial apoptotic pathway, which showed cardioprotective properties in a murine model of AMI after a single bolus of intravenous administration. More importantly, similar peptidic strategies and tools are likely to be adaptable to many other situations in which cells have to be protected from apoptosis such as stroke or organ transplantation.