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Special Issue "Cell-penetrating Peptides 2012"

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A special issue of Pharmaceuticals (ISSN 1424-8247).

Deadline for manuscript submissions: closed (30 September 2012)

Special Issue Editor

Guest Editor
Prof. Dr. Vladimir P. Torchilin (Website)

University Distinguished Professor, Director, Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, 140 The Fenway, room 214, 360 Huntington Avenue Boston, MA 02115, USA
Fax: +617 373 7509
Interests: drug carriers; drug delivery sytems; drug targeting; liposomes; micelles; experimental cancer immunology; imaging agents

Special Issue Information

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

Keywords

  • intracellular delivery
  • cell-penetrating peptides
  • protein transduction domains
  • drug targeting
  • pharmaceutical nanocarriers

Published Papers (23 papers)

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Research

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Open AccessArticle Cell Penetration Properties of a Highly Efficient Mini Maurocalcine Peptide
Pharmaceuticals 2013, 6(3), 320-339; doi:10.3390/ph6030320
Received: 23 February 2013 / Revised: 6 March 2013 / Accepted: 7 March 2013 / Published: 18 March 2013
Cited by 7 | PDF Full-text (1300 KB) | HTML Full-text | XML Full-text
Abstract
Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of [...] Read more.
Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessArticle Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells
Pharmaceuticals 2013, 6(2), 184-203; doi:10.3390/ph6020184
Received: 1 November 2012 / Revised: 29 January 2013 / Accepted: 30 January 2013 / Published: 6 February 2013
Cited by 7 | PDF Full-text (2068 KB) | HTML Full-text | XML Full-text
Abstract
Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing [...] Read more.
Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles
Pharmaceuticals 2013, 6(2), 204-222; doi:10.3390/ph6020204
Received: 19 November 2012 / Revised: 22 January 2013 / Accepted: 23 January 2013 / Published: 6 February 2013
Cited by 5 | PDF Full-text (2818 KB) | HTML Full-text | XML Full-text
Abstract
Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to [...] Read more.
Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans
Pharmaceuticals 2012, 5(12), 1265-1281; doi:10.3390/ph5121265
Received: 10 October 2012 / Revised: 13 November 2012 / Accepted: 14 November 2012 / Published: 23 November 2012
Cited by 6 | PDF Full-text (1121 KB) | HTML Full-text | XML Full-text
Abstract
Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used [...] Read more.
Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Intranasal Delivery of Camptothecin-Loaded Tat-Modified Nanomicells for Treatment of Intracranial Brain Tumors
Pharmaceuticals 2012, 5(10), 1092-1102; doi:10.3390/ph5101092
Received: 3 September 2012 / Revised: 27 September 2012 / Accepted: 9 October 2012 / Published: 15 October 2012
Cited by 11 | PDF Full-text (321 KB) | HTML Full-text | XML Full-text
Abstract
The blood-brain barrier is a substantial obstacle for delivering anticancer agents to brain tumors, and new strategies for bypassing it are sorely needed for brain tumor therapy. Intranasal delivery provides a practical, noninvasive method for delivering therapeutic agents to the brain. Intranasal [...] Read more.
The blood-brain barrier is a substantial obstacle for delivering anticancer agents to brain tumors, and new strategies for bypassing it are sorely needed for brain tumor therapy. Intranasal delivery provides a practical, noninvasive method for delivering therapeutic agents to the brain. Intranasal application of nano-sized micelles that have been modified with Tat peptide facilitates brain delivery of fluorescent model materials. In this study, we evaluated a nose-to-brain delivery system for brain tumor therapy. We nasally administered the anti-tumor drug camptothecin (CPT) in solution and in methoxy poly(ethylene glycol) (MPEG)/poly(e-caprolactone) (PCL) amphiphilic block copolymers (MPEG-PCL) and cell penetrating peptide, Tat analog-modified MPEG-PCL (MPEG-PCL-Tat) MPEG-PCL-Tat to rats bearing intracranial glioma tumors and quantified the cytotoxicity against glioma cells, and the therapeutic effects. CPT-loaded MPEG-PCL-Tat micelles showed higher cytotoxicity than CPT-loaded MPEG-PCL. CPT-free MPEG-PCL-Tat didn’t show any cytotoxicity, even at high concentrations (2 mmol/mL). CPT-loaded MPEG-PCL-Tat micelles significantly prolonged the median survival of rats. These results indicate that intranasal delivery of anti-cancer drugs with cell penetrating peptide-modified nanomicelles might be an effective therapy for brain tumors. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity
Pharmaceuticals 2010, 3(12), 3594-3613; doi:10.3390/ph3123594
Received: 17 November 2010 / Accepted: 1 December 2010 / Published: 15 December 2010
Cited by 18 | PDF Full-text (2972 KB) | HTML Full-text | XML Full-text
Abstract
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, [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration
Pharmaceuticals 2010, 3(4), 1232-1240; doi:10.3390/ph3041232
Received: 11 February 2010 / Revised: 16 April 2010 / Accepted: 21 April 2010 / Published: 23 April 2010
PDF Full-text (640 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessArticle Imperatoxin A, a Cell-Penetrating Peptide from Scorpion Venom, as a Probe of Ca2+-Release Channels/Ryanodine Receptors
Pharmaceuticals 2010, 3(4), 1093-1107; doi:10.3390/ph3041093
Received: 18 March 2010 / Accepted: 11 April 2010 / Published: 13 April 2010
Cited by 12 | PDF Full-text (1216 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessArticle Cell-Penetrating Peptides: A Comparative Study on Lipid Affinity and Cargo Delivery Properties
Pharmaceuticals 2010, 3(4), 1045-1062; doi:10.3390/ph3041045
Received: 22 December 2009 / Revised: 23 March 2010 / Accepted: 29 March 2010 / Published: 30 March 2010
Cited by 9 | PDF Full-text (399 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material
Pharmaceuticals 2010, 3(3), 621-635; doi:10.3390/ph3030621
Received: 28 December 2009 / Revised: 5 March 2010 / Accepted: 9 March 2010 / Published: 12 March 2010
PDF Full-text (431 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessArticle Comparison of Functional Protein Transduction Domains Using the NEMO Binding Domain Peptide
Pharmaceuticals 2010, 3(1), 110-124; doi:10.3390/ph3010110
Received: 24 October 2009 / Revised: 22 December 2009 / Accepted: 24 December 2009 / Published: 8 January 2010
Cited by 3 | PDF Full-text (1440 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessArticle Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity
Pharmaceuticals 2009, 2(2), 49-65; doi:10.3390/ph2020049
Received: 10 August 2009 / Revised: 22 September 2009 / Accepted: 24 September 2009 / Published: 25 September 2009
Cited by 26 | PDF Full-text (513 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Review

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Open AccessReview Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges
Pharmaceuticals 2012, 5(11), 1177-1209; doi:10.3390/ph5111177
Received: 8 October 2012 / Revised: 25 October 2012 / Accepted: 26 October 2012 / Published: 1 November 2012
Cited by 69 | PDF Full-text (526 KB) | HTML Full-text | XML Full-text
Abstract
Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this [...] Read more.
Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessReview Cell-Penetrating Recombinant Peptides for Potential Use in Agricultural Pest Control Applications
Pharmaceuticals 2012, 5(10), 1054-1063; doi:10.3390/ph5101054
Received: 27 August 2012 / Revised: 21 September 2012 / Accepted: 21 September 2012 / Published: 28 September 2012
Cited by 3 | PDF Full-text (214 KB) | HTML Full-text | XML Full-text
Abstract
Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these [...] Read more.
Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies
Pharmaceuticals 2012, 5(9), 991-1007; doi:10.3390/ph5090991
Received: 21 August 2012 / Revised: 30 August 2012 / Accepted: 6 September 2012 / Published: 12 September 2012
Cited by 21 | PDF Full-text (256 KB) | HTML Full-text | XML Full-text
Abstract
Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either [...] Read more.
Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either of conventional small molecular drugs or oligonucleotide based therapeutics. This review is focused on the cancer applications of cell penetrating peptides as delivery systems; different aspects of drug loading, cargoes and delivery are discussed together with methods for targeted delivery, activatable cell-penetrating peptides and transducible agents coupled to cell-penetrating peptides. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Spotlight on Human LL-37, an Immunomodulatory Peptide with Promising Cell-Penetrating Properties
Pharmaceuticals 2010, 3(11), 3435-3460; doi:10.3390/ph3113435
Received: 4 October 2010 / Revised: 23 October 2010 / Accepted: 29 October 2010 / Published: 1 November 2010
Cited by 7 | PDF Full-text (948 KB) | HTML Full-text | XML Full-text
Abstract
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, [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Building Cell Selectivity into CPP-Mediated Strategies
Pharmaceuticals 2010, 3(5), 1456-1490; doi:10.3390/ph3051456
Received: 8 January 2010 / Revised: 29 April 2010 / Accepted: 5 May 2010 / Published: 14 May 2010
Cited by 19 | PDF Full-text (1368 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessReview Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems
Pharmaceuticals 2010, 3(4), 961-993; doi:10.3390/ph3040961
Received: 22 December 2009 / Revised: 20 February 2010 / Accepted: 29 March 2010 / Published: 30 March 2010
Cited by 84 | PDF Full-text (330 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Dendritic Guanidines as Efficient Analogues of Cell Penetrating Peptides
Pharmaceuticals 2010, 3(3), 636-666; doi:10.3390/ph3030636
Received: 18 December 2010 / Revised: 10 February 2010 / Accepted: 9 March 2010 / Published: 12 March 2010
Cited by 16 | PDF Full-text (398 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Open AccessReview Targeting the Tumour: Cell Penetrating Peptides for Molecular Imaging and Radiotherapy
Pharmaceuticals 2010, 3(3), 600-620; doi:10.3390/ph3030600
Received: 26 December 2009 / Revised: 2 February 2010 / Accepted: 10 March 2010 / Published: 11 March 2010
Cited by 14 | PDF Full-text (444 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Cell-Penetrating Peptides for Antiviral Drug Development
Pharmaceuticals 2010, 3(3), 448-470; doi:10.3390/ph3030448
Received: 24 December 2009 / Revised: 6 February 2010 / Accepted: 1 March 2010 / Published: 2 March 2010
Cited by 10 | PDF Full-text (525 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
Open AccessReview Cell Permeable Peptides: A Promising Tool to Deliver Neuroprotective Agents in the Brain
Pharmaceuticals 2010, 3(2), 379-392; doi:10.3390/ph3020379
Received: 18 December 2008 / Revised: 20 January 2010 / Accepted: 28 January 2010 / Published: 3 February 2010
Cited by 3 | PDF Full-text (175 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)

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Open AccessConcept Paper The Potential Role of Cell Penetrating Peptides in the Intracellular Delivery of Proteins for Therapy of Erythroid Related Disorders
Pharmaceuticals 2013, 6(1), 32-53; doi:10.3390/ph6010032
Received: 31 October 2012 / Revised: 20 December 2012 / Accepted: 27 December 2012 / Published: 7 January 2013
PDF Full-text (223 KB) | HTML Full-text | XML Full-text
Abstract
The erythroid related disorders (ERDs) represent a large group of hematological diseases, which in most cases are attributed either to the deficiency or malfunction of biosynthetic enzymes or oxygen transport proteins. Current treatments for these disorders include histo-compatible erythrocyte transfusions or allogeneic [...] Read more.
The erythroid related disorders (ERDs) represent a large group of hematological diseases, which in most cases are attributed either to the deficiency or malfunction of biosynthetic enzymes or oxygen transport proteins. Current treatments for these disorders include histo-compatible erythrocyte transfusions or allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy delivered via suitable viral vectors or genetically modified HSCs have been under way. Protein Transduction Domain (PTD) technology has allowed the production and intracellular delivery of recombinant therapeutic proteins, bearing Cell Penetrating Peptides (CPPs), into a variety of mammalian cells. Remarkable progress in the field of protein transduction leads to the development of novel protein therapeutics (CPP-mediated PTs) for the treatment of monogenetic and/or metabolic disorders. The “concept” developed in this paper is the intracellular protein delivery made possible via the PTD technology as a novel therapeutic intervention for treatment of ERDs. This can be achieved via four stages including: (i) the production of genetically engineered human CPP-mediated PT of interest, since the corresponding native protein either is missing or is mutated in the erythroid progenitor cell (ErPCs) or mature erythrocytes of patients; (ii) isolation of target cells from the peripheral blood of the selected patients; (iii) ex vivo transduction of cells with the CPP-mediated PT of interest; and (iv) re-administration of the successfully transduced cells back into the same patients. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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