http://www.mdpi.com/journal/pharmaceuticals/special_issues/peptides/ Dear Colleagues, Intracellular delivery of therapeutic molecules is one of the key problems in drug delivery in general. Many pharmaceutical agents should be delivered intracellularly to exert their therapeutic action inside cytoplasm or onto individual organelles, such as nuclei, lysosomes, or mitochondria. However, cell membranes prevent proteins, peptides, and nanoparticulate drug carriers from entering cells. So far, multiple and only partially successful attempts have been made to bring various drugs and drug-loaded pharmaceutical carriers directly into the cell cytoplasm bypassing the endocytic pathway, to protect drugs and DNA from the lysosomal degradation. A promising approach that seems to be the solution of overcoming the cellular barrier for intracellular drug delivery has emerged over the last decade. In this approach, certain proteins or peptides can be tethered to the hydrophilic drug of interest and together the construct possesses the ability to translocate across the plasma membrane and deliver the payload intracellularly; the process termed as “protein transduction”. Such proteins or peptides contain domains of less than 20 amino acids, Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs) that are highly rich in basic residues. These peptides have been used for intracellular delivery of various cargoes with molecular weights significantly greater than their own. This special issue will be dealing with pharmaceutical application of cell-penetrating peptides – a challenging and promising area in pharmaceutical research. Prof. Dr. Vladimir P. Torchilin Guest Editor {snippet name="submission_info"} http://www.mdpi.com/1424-8247/3/12/3594/ Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cell-penetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-b-cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycan-deficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 mM when the cells were cultured in medium containing 1 mM – 1.6 mM VPTLK. To determine the protein-transduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus of Cre, and these fusion proteins were added to the culture medium of Tex-LoxP EG cells. Both VPTLK-Cre and KLPVM-Cre were able to turn on GFP expression in these cells, suggesting that CPP5s have protein-transduction activity. Since CPP5s have very low cytotoxic activity, even at a concentration of 1.6 mM in the medium, CPP5s could be utilized as a new tool for drug delivery into cells. http://www.mdpi.com/1424-8247/3/12/3594/ Wed, 15 Dec 2010 00:00:00 CET Pharmaceuticals 2010-12-15 3 12 Article 3594 3613 1424-8247 Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity 2010-12-15 doi: 10.3390/ph3123594 Jose A. Gomez Joseph Chen Justine Ngo Dagmar Hajkova I-Ju Yeh Vivian Gama Masaru Miyagi Shigemi Matsuyama http://www.mdpi.com/1424-8247/3/11/3435/ Cationic antimicrobial peptides are major components of innate immunity and help control the initial steps of the infectious process. They are expressed not only by immunocytes, but also by epithelial cells. They share an amphipathic secondary structure with a polar cationic site, which explains their tropism for prokaryote membranes and their hydrophobic site contributing to the destructuration of these membranes. LL-37 is the only cationic antimicrobial peptide derived from human cathelicidin. LL-37 can also cross the plasma membrane of eukaryotic cells, probably through special domains of this membrane called lipid rafts. This transfer could be beneficial in the context of vaccination: the activation of intracellular toll-like receptors by a complex formed between CpG oligonucleotides and LL-37 could conceivably play a major role in the building of a cellular immunity involving NK cells. http://www.mdpi.com/1424-8247/3/11/3435/ Mon, 01 Nov 2010 00:00:00 CET Pharmaceuticals 2010-11-01 3 11 Review 3435 3460 1424-8247 Spotlight on Human LL-37, an Immunomodulatory Peptide with Promising Cell-Penetrating Properties 2010-11-01 doi: 10.3390/ph3113435 Michèle Seil Carole Nagant Jean-Paul Dehaye Michel Vandenbranden Marc Ferdinand Lensink http://www.mdpi.com/1424-8247/3/5/1456/ There is a pressing need for more effective and selective therapies for cancer and other diseases. Consequently, much effort is being devoted to the development of alternative experimental approaches based on selective systems, which are designed to be specifically directed against target cells. In addition, a large number of highly potent therapeutic molecules are being discovered. However, they do not reach clinical trials because of their low delivery, poor specificity or their incapacity to bypass the plasma membrane. Cell-penetrating peptides (CPPs) are an open door for cell-impermeable compounds to reach intracellular targets. Putting all these together, research is sailing in the direction of the design of systems with the capacity to transport new drugs into a target cell. Some CPPs show cell type specificity while others require modifications or form part of more sophisticated drug delivery systems. In this review article we summarize several strategies for directed drug delivery involving CPPs that have been reported in the literature. http://www.mdpi.com/1424-8247/3/5/1456/ Fri, 14 May 2010 00:00:00 CEST Pharmaceuticals 2010-05-14 3 5 Review 1456 1490 1424-8247 Building Cell Selectivity into CPP-Mediated Strategies 2010-05-14 doi: 10.3390/ph3051456 Martín Teixidó Giralt http://www.mdpi.com/1424-8247/3/4/1232/ Cdk5 is essential for neuronal differentiation processes in the brain. Activation of Cdk5 requires the association with the mostly neuron-specific p35 or p39. Overactivation of CDK5 by cleavage of p35 into p25 is thought to be involved in neurodegenerative processes. Here, we have tested an approach to inhibit pathological Cdk5 activation with a Tat-linked dominant-negative fragment of p25. It reduced cell death induced by staurosporine and showed a tendency to alleviate manganese-induced cell death, while it did not protect against 6-OHDA toxicity. Our results suggest that the Tat technique is a suitable tool to inhibit dysregulated CDK5. http://www.mdpi.com/1424-8247/3/4/1232/ Fri, 23 Apr 2010 00:00:00 CEST Pharmaceuticals 2010-04-23 3 4 Article 1232 1240 1424-8247 Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration 2010-04-23 doi: 10.3390/ph3041232 Liman Weishaupt Bähr Dietz http://www.mdpi.com/1424-8247/3/4/1093/ Scorpion venoms are rich in ion channel-modifying peptides, which have proven to be invaluable probes of ion channel structure-function relationship. We previously isolated imperatoxin A (IpTxa), a 3.7 kDa peptide activator of Ca2+-release channels/ryanodine receptors (RyRs) [1,2,3] and founding member of the calcin family of scorpion peptides. IpTxa folds into a compact, mostly hydrophobic molecule with a cluster of positively-charged, basic residues polarized on one side of the molecule that possibly interacts with the phospholipids of cell membranes. To investigate whether IpTxa permeates external cellular membranes and targets RyRs in vivo, we perfused IpTxa on intact cardiomyocytes while recording field-stimulated intracellular Ca2+ transients. To further investigate the cell-penetrating capabilities of the toxin, we prepared thiolated, fluorescent derivatives of IpTxa. Biological activity and spectroscopic properties indicate that these derivatives retain high affinity for RyRs and are only 5- to 10-fold less active than native IpTxa. Our results demonstrate that IpTxa is capable of crossing cell membranes to alter the release of Ca2+ in vivo, and has the capacity to carry a large, membrane-impermeable cargo across the plasma membrane, a finding with exciting implications for novel drug delivery. http://www.mdpi.com/1424-8247/3/4/1093/ Tue, 13 Apr 2010 00:00:00 CEST Pharmaceuticals 2010-04-13 3 4 Article 1093 1107 1424-8247 Imperatoxin A, a Cell-Penetrating Peptide from Scorpion Venom, as a Probe of Ca2+-Release Channels/Ryanodine Receptors 2010-04-13 doi: 10.3390/ph3041093 Gurrola Capes Zamudio Possani Valdivia http://www.mdpi.com/1424-8247/3/4/1045/ A growing number of natural and/or synthetic peptides with cell membrane penetrating capability have been identified and described in the past years. These molecules have been considered promising tools for delivering bioactive compounds into various cell types. Although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. In this work we try to connect the capability to interact with model lipid membrane and structural and chemical characteristics of CPPs in order to obtain a biophysical classification that predicts the behavior of CPP-cargo molecules in cell systems. Indeed, the binding with cell membrane is one of the primary step in the interaction of CPPs with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how CPPs may translocate a membrane without destroying it and how this interactions come into play in shuttling CPPs via different routes with different efficiency. We analyzed by CD and fluorescence spectroscopies the binding properties of six different CPPs (kFGF, Nle54-Antp and Tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues) in absence or presence of the same cargo peptide (the 392-401pTyr396 fragment of HS1 protein). The phospholipid binding properties were correlated to the conformational and chemical characteristics of peptides, as well as to the cell penetrating properties of the CPP-cargo conjugates. Results show that even if certain physico-chemical properties (conformation, positive charge) govern CPP capability to interact with the model membrane, these cannot fully explain cell-permeability properties. http://www.mdpi.com/1424-8247/3/4/1045/ Tue, 30 Mar 2010 00:00:00 CEST Pharmaceuticals 2010-03-30 3 4 Article 1045 1062 1424-8247 Cell-Penetrating Peptides: A Comparative Study on Lipid Affinity and Cargo Delivery Properties 2010-03-30 doi: 10.3390/ph3041045 Ruzza Biondi Marchiani Antolini Calderan http://www.mdpi.com/1424-8247/3/4/961/ The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery. http://www.mdpi.com/1424-8247/3/4/961/ Tue, 30 Mar 2010 00:00:00 CEST Pharmaceuticals 2010-03-30 3 4 Review 961 993 1424-8247 Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems 2010-03-30 doi: 10.3390/ph3040961 Trabulo Cardoso Mano De Lima http://www.mdpi.com/1424-8247/3/3/636/ The widespread application of cell penetrating agents to clinical therapeutics and imaging agents relies on the ability to prepare them on a large scale and to readily conjugate them to their cargos. Dendritic analogues of cell penetrating peptides, with multiple guanidine groups on their peripheries offer advantages as their high symmetry allows them to be efficiently synthesized, while orthogonal functionalities at their focal points allow them to be conjugated to cargo using simple synthetic methods. Their chemical structures and properties are also highly tunable as their flexibility and the number of guanidine groups can be tuned by altering the dendritic backbone or the linkages to the guanidine groups. This review describes the development of cell-penetrating dendrimers based on several different backbones, their structure-property relationships, and comparisons of their efficacies with those of known cell penetrating peptides. The toxicities of these dendritic guanidines are also reported as well as their application towards the intracellular delivery of biologically significant cargos including proteins and nanoparticles. http://www.mdpi.com/1424-8247/3/3/636/ Fri, 12 Mar 2010 00:00:00 CET Pharmaceuticals 2010-03-12 3 3 Review 636 666 1424-8247 Dendritic Guanidines as Efficient Analogues of Cell Penetrating Peptides 2010-03-12 doi: 10.3390/ph3030636 Bonduelle Gillies http://www.mdpi.com/1424-8247/3/3/621/ Interest in cell-penetrating peptides (CPPs) as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue) for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin--galactosidase (ABG), which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases. http://www.mdpi.com/1424-8247/3/3/621/ Fri, 12 Mar 2010 00:00:00 CET Pharmaceuticals 2010-03-12 3 3 Article 621 635 1424-8247 Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material 2010-03-12 doi: 10.3390/ph3030621 Saar Saar Hansen Langel Pooga http://www.mdpi.com/1424-8247/3/3/600/ Over the last couple of years, the number of original papers and reviews discussing various applications of cell penetrating peptides (CPPs) has grown exponentially. This is not remarkable since CPPs are capable of transporting the most varying cargo across cell membranes which is one of the biggest problems in drug delivery and targeted therapy. In this review, we focus on the use of CPPs and related peptides for delivery of imaging contrast agents and radionuclides to cells and tissues with the ultimate goal of in vivo molecular imaging and molecular radiotherapy of intracellular and even intranuclear targets. http://www.mdpi.com/1424-8247/3/3/600/ Thu, 11 Mar 2010 00:00:00 CET Pharmaceuticals 2010-03-11 3 3 Review 600 620 1424-8247 Targeting the Tumour: Cell Penetrating Peptides for Molecular Imaging and Radiotherapy 2010-03-11 doi: 10.3390/ph3030600 Veerle Kersemans Bart Cornelissen http://www.mdpi.com/1424-8247/3/3/448/ Viral diseases affect hundreds of millions of people worldwide, and the few available drugs to treat these diseases often come with limitations. The key obstacle to the development of new antiviral agents is their delivery into infected cells in vivo. Cell-penetrating peptides (CPPs) are short peptides that can cross the cellular lipid bilayer with the remarkable capability to shuttle conjugated cargoes into cells. CPPs have been successfully utilized to enhance the cellular uptake and intracellular trafficking of antiviral molecules, and thereby increase the inhibitory activity of potential antiviral proteins and oligonucleotide analogues, both in cultured cells and in animal models. This review will address the notable findings of these studies, highlighting some promising results and discussing the challenges CPP technology has to overcome for further clinical applications. http://www.mdpi.com/1424-8247/3/3/448/ Tue, 02 Mar 2010 00:00:00 CET Pharmaceuticals 2010-03-02 3 3 Review 448 470 1424-8247 Cell-Penetrating Peptides for Antiviral Drug Development 2010-03-02 doi: 10.3390/ph3030448 Melaine Delcroix Lee W. Riley http://www.mdpi.com/1424-8247/3/2/379/ The inability of most drugs to cross the blood-brain barrier and/or plasma membrane limits their use for biomedical applications in the brain. Cell Permeable Peptides (CPPs) overcome this problem and are effective in vivo, crossing the plasma membrane and the blood-brain barrier. CPPs deliver a wide variety of compounds intracellularly in an active form. In fact, many bioactive cargoes have neuroprotective properties, and due to their ability to block protein-protein interactions, offer exciting perspectives in the clinical setting. In this review we give an overview of the Cell Permeable Peptides strategy to deliver neuroprotectants against neurodegeneration in the CNS. http://www.mdpi.com/1424-8247/3/2/379/ Wed, 03 Feb 2010 00:00:00 CET Pharmaceuticals 2010-02-03 3 2 Review 379 392 1424-8247 Cell Permeable Peptides: A Promising Tool to Deliver Neuroprotective Agents in the Brain 2010-02-03 doi: 10.3390/ph3020379 Xanthi Antoniou Tiziana Borsello http://www.mdpi.com/1424-8247/3/1/110/ Protein transduction domains (PTDs), both naturally occurring and synthetic, have been extensively utilized for intracellular delivery of biologically active molecules both in vitro and in vivo. However, most comparisons of transduction efficiency have been performed using fluorescent markers. To compare efficiency of functional protein transduction, a peptide derived from IkB kinase ß (IKKß) that prevents formation of an active IKK complex was used as a biologically active cargo. This peptide, termed NEMO Binding Domain (NBD), is able to block activation of the transcriptional factor NF-κB by IKK, but not basal NF-κB activity. Our results demonstrate that Antp and Tat PTDs were most effective for delivery of NBD for inhibition of NF-kB activation compared to other PTD-NBD in both Hela and 293 cells, however, at higher concentrations (100 µM), the Antp-NBD as well as the FGF-NBD peptide caused significant cellular toxicity. In contrast to the cell culture results, delivery of NBD using 8K (octalysine) and 6R (six arginine) were the most effect in blocking inflammation following local, footpad delivery in a KLH-induced DTH murine model of inflammatory arthritis. These results demonstrate differences between PTDs for delivery of a functional cargo between cell types. http://www.mdpi.com/1424-8247/3/1/110/ Fri, 08 Jan 2010 00:00:00 CET Pharmaceuticals 2010-01-08 3 1 Article 110 124 1424-8247 Comparison of Functional Protein Transduction Domains Using the NEMO Binding Domain Peptide 2010-01-08 doi: 10.3390/ph3010110 Khaleel Khaja Paul Robbins http://www.mdpi.com/1424-8247/2/2/49/ Cell-penetrating peptides (CPP) have become a widely used tool for efficient cargo delivery into cells. However, one limiting fact is their uptake by endocytosis causing the enclosure of the CPP-cargo construct within endosomes. One often used method to enhance the outflow into the cytosol is the fusion of endosome-disruptive peptide or protein sequences to CPP. But, until now, no studies exist investigating the effects of the fusion peptide to the cellular distribution, structural arrangements and cytotoxic behaviour of the CPP. In this study, we attached a short modified sequence of hemagglutinin subunit HA2 to different CPP and analysed the biologic activity of the new designed peptides. Interestingly, we observed an increased cytosolic distribution but also highly toxic activities in the micromolar range against several cell lines. Structural analysis revealed that attachment of the fusion peptide had profound implications on the whole conformation of the peptide, which might be responsible for membrane interaction and endosome disruption. http://www.mdpi.com/1424-8247/2/2/49/ Fri, 25 Sep 2009 00:00:00 CEST Pharmaceuticals 2009-09-25 2 2 Article 49 65 1424-8247 Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity 2009-09-25 doi: 10.3390/ph2020049 Ines Neundorf Robert Rennert Jan Hoyer Franziska Schramm Kristin Löbner Igor Kitanovic Stefan Wölfl