Search

The cRGD-conjugated Aib-containing amphipathic helical peptide, MAP(Aib) derivative (PI), has been reported to be a useful carrier for siRNA delivery into cells. We have conducted a series of structure-activity relationship studies of the influence of the balance between hydrophobicity and basicity on the amphipathicity of PI , and synthesized peptides having a larger number of Lys residues than PI. Increasing the number of basic residues in the amphipathic helix suppressed the ability to deliver siRNA into cells. It was concluded that the balance between hydrophobicity and basicity in the PI helix was important for siRNA delivery into cells. Furthermore, the siRNA delivering ability of PI was specific to cancer cells, such as A549, U-87 MG, and WiDr cells, and was low in normal cells, namely, NIH3T3 cells. Next, we examined the potential of PI as a carrier for the delivery of microRNA-133b (miR-133b), which is known to be an anti-oncomiR. PI enhanced the delivery of miR-133b into WiDr cells, which resulted in the suppression of endogenous protein expression. [ABSTRACT FROM AUTHOR]

The lung remains an attractive target for the gene therapy of monogenetic diseases such as cystic fibrosis (CF). Despite over 27 clinical trials, there are still very few gene therapy vectors that have shown any improvement in lung function; highlighting the need to develop formulations with improved gene transfer potency and the desirable physiochemical characteristics for efficacious therapy. Herein, we introduce a novel cell penetrating peptide (CPP)-based non-viral vector that utilises glycosaminoglycan (GAG)-binding enhanced transduction (GET) for highly efficient gene transfer. GET peptides couple directly with DNA through electrostatic interactions to form nanoparticles (NPs). In order to adapt the GET peptide for efficient in vivo delivery, we engineered PEGylated versions of the peptide and employed a strategy to form DNA NPs with different densities of PEG coatings. We were able to identify candidate formulations (PEGylation rates ≥40%) that shielded the positively charged surface of particles, maintained colloidal stability in bronchoalveolar lavage fluid (BALF) and retained gene transfer activity in human bronchial epithelial cell lines and precision cut lung slices (PCLS) in vitro. Using multiple particle tracking (MPT) technology, we demonstrated that PEG-GET complexes were able to navigate the mucus mesh and diffuse rapidly through patient CF sputum samples ex vivo. When tested in mouse lung models in vivo, PEGylated particles demonstrated superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid compared to non-PEGylated complexes. Furthermore, gene expression was significantly enhanced in comparison to polyethylenimine (PEI), a non-viral gene carrier that has been widely tested in pre-clinical settings. This work describes an innovative approach that combines novel GET peptides for enhanced transfection with a tuneable PEG coating for efficacious lung gene therapy. [ABSTRACT FROM AUTHOR]

Cellular reprogramming is a promising technology in regenerative medicine, but most studies have been performed by using expression vectors. For future clinical applications, it is necessary to establish a system in which cell engineering can be manipulated without any risk of damaging the genome. Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11). We named the peptide “nuclear trafficking peptide” (NTP) and applied it to a protein-based artificial transcription factor (NTP-ATF), which was composed of a transcription activator-like effector and transcription domain (VP64). An NTP-ATF designed to the proximal promoter region of the microRNA-302/367 cluster efficiently induced endogenous RNA expression at an extremely low concentration (0.25 nM), and repetitive treatment of mouse embryonic fibroblasts with NTP-ATF generated induced pluripotent stem-like cells, which gave chimeric mice. Together with the observation that recombinant NTP-ATF protein did not induce any apparent cytotoxicity, we propose that NTP-ATF is a promising system for cellular reprogramming applicable to regenerative medicine. [ABSTRACT FROM AUTHOR]

Herein, a smart pH-sensitive nanoparticle (DGL-PEG-Tat-KK-DMA-DOX) was prepared to achieve the selective intracellular drug delivery. In this nanoparticle, a PEG-grafted cell penetrating peptide (PEG-Tat-KK) was designed and acted as the cell penetrating segment. By introducing the pH-sensitive amide bonds between the peptide and blocking agent (2,3-dimethylmaleic anhydride, DMA), the controllable moiety (PEG-Tat-KK-DMA) endowed the nanoparticle with a charge-switchable shell and temporarily blocked penetrating function, thus improving the specific internalization. Besides, dendrigraft poly-L-lysine (DGL) used as the skeleton can greatly improve the drug loading because of the highly dendritic framework. Under the stimuli of acidic pH, this nanoparticle exhibited a remarkable charge-switchable property. The drug release showed an expected behavior with little release in the neutral pH media but relatively fast release in the acidic media. The in vitro experiments revealed that the cellular uptake and cytotoxicity were significantly enhanced after the pH was decreased. In vivo biodistribution and antitumor research indicated that the nanoparticle had noteworthy specificity and antitumor efficacy with a tumor inhibition rate of 79.7%. These results verified this nanoparticle could efficiently improve the selective intracellular delivery and possessed a great potential in tumor treatment. [ABSTRACT FROM AUTHOR]

The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp- d -Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide ( PI ) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure–activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus ( PII ) and both the N- and C-termini ( PIII ) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 ( PIV ), 12 ( PV ), and 8 ( PVI ) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII , as well as PI , could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) have been widely used to deliver nucleic acid molecules. Generally, CPPs consisting of short amino acid sequences have a linear structure, resulting in a weak complexation and low transfection efficacy. To overcome these drawbacks, a novel type of CPP is required to enhance the delivery efficacy while maintaining its safe use at the same time. Herein, we report that a bioreducible branched poly-CPP structure capable of responding to reducing conditions attained both outstanding delivery effectiveness and selective gene release in carcinoma cells. Branched structures provide unusually strong electrostatic attraction between DNA and siRNA molecules, thereby improving the transfection capability through a tightly condensed form. We designed a modified type of nona-arginine (mR9) and synthesized a branched-mR9 (B-mR9) using disulfide bonds. A novel B-mR9/pDNA polyplex exhibited redox-cleavability and high transfection efficacy compared to conventional CPPs, with higher cell viability as well. B-mR9/VEGF siRNA polyplex exhibited significant serum stability and high gene-silencing effects in vitro . Furthermore, the B-mR9 polyplex showed outstanding tumor accumulation and inhibition ability in vivo . The results suggest that the bioreducible branched poly CPP has great potential as a gene delivery platform. [ABSTRACT FROM AUTHOR]

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp- d -Phe-Cys-)], which is known to bind to α V β 3 integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond ( PI ) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds ( PIII ) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI /siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII /siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI /siRNA complex might involve specific interactions of the RGD group with the α V β 3 integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI /10 nM siRNA. In contrast, the PIII /siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII /siRNA complex. Considering that the cellular uptake of the PIII /siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH 2 ). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH 2 ) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH 2 ) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132 h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields. [ABSTRACT FROM AUTHOR]

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis , gradual dose-dependent single exposure pediocin-resistant (Ped r ) mutants of E . faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Ped r mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in Ped r E . faecalis . Decreased level of branched chain fatty acids in Ped r mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Ped r mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Ped r mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC 50 of CAMPs for wild-type and Ped r mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs. [ABSTRACT FROM AUTHOR]

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this "Pep-c19" also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs. [ABSTRACT FROM AUTHOR]

Abstract: During the last decade peptoid-based molecular transporters have been broadly applied. They are highly valued for their easy synthesis and their superior stability against enzymatic degradation. The special structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating properties. [Copyright &y& Elsevier]

Abstract: Background: Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl–CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process. Methods: In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles. Results: The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence. Conclusions: These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently. General significance: Understanding the mechanism behind Fl–CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds. [Copyright &y& Elsevier]

Abstract: A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells. [Copyright &y& Elsevier]

Highlights: [•] We synthesized carboxylated and PEGylated bifunctionalized InP/ZnS QDs (QInP). [•] Cell-penetrating peptides (CPPs) could deliver QInP into human A549 cells. [•] Five hundred nM of CPP/QInP and QInP at or below 1μM did not reduce cell viability. [•] QInP are biocompatible nanoparticles for use in biomedical applications. [ABSTRACT FROM AUTHOR]

Abstract: Cell-penetrating peptide (CPP)-mediated intracellular drug delivery system, often specifically termed as “the Trojan horse approach”, has become the “holy grail” in achieving effective delivery of macromolecular compounds such as proteins, DNA, siRNAs, and drug carriers. It is characterized by the unique cell- (or receptor-), temperature-, and payload-independent mechanisms, therefore offering potent means to improve poor cellular uptake of a variety of macromolecular drugs. Nevertheless, this “Trojan horse” approach also acts like a double-edged sword, causing serious safety and toxicity concerns to normal tissues or organs for in vivo application, due to lack of target selectivity of the powerful cell penetrating activity. To overcome this problem of potent yet non-selective penetration vs. targeting delivery, a number of “smart” strategies have been developed in recent years, including controllable CPP-based drug delivery systems based on various stimuli-responsive mechanisms. This review article provides a fundamental understanding of these smart systems, as well as a discussion of their real-time in vivo applicability. [Copyright &y& Elsevier]

Abstract: Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes. [Copyright &y& Elsevier]

Highlights: [•] AMPs are multifunctional molecules that selectively permeate microbial membranes. [•] Negative Gaussian curvature (NGC) enables many types of membrane permeation. [•] NGC generation by AMPs constrains their compositions in the form of a general rule. [•] We show that the sequences of known cationic AMPs are consistent with this rule. [ABSTRACT FROM AUTHOR]

Abstract: For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms. [Copyright &y& Elsevier]

Abstract: Cell-penetrating peptides (CPPs) are a class of short, often cationic peptides that have the capability to translocate across cellular membranes, and although the translocation most likely involves several pathways, they interact directly with membranes, as well as with model bilayers. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes it is of great importance to investigate CPPs at atomic resolution in a suitable membrane model. Moreover, the location in bilayers is likely to be correlated with the translocation mechanism. Solution-state NMR offers a unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating CPP–lipid interactions. Structural propensities and cell-penetrating capabilities can be derived from a combination of CPP solution structures and studies of the effect that the peptides have on bilayers and the localization in a bilayer. [Copyright &y& Elsevier]

Abstract: Cell penetrating peptides (CPPs) are usually short, highly cationic peptides that are capable of crossing the cell membrane and transport cargos of varied size and nature in cells by energy- and receptor-independent mechanisms. An additional potential is the newly discovered anti-tumor activity of certain CPPs, including RW16 (RRWRRWWRRWWRRWRR) which is derived from penetratin and is investigated here. The use of CPPs in therapeutics, diagnosis and potential application as anti-tumor agents increases the necessity of understanding their mode of action, a subject yet not totally understood. With this in mind, the membrane interaction and perturbation mechanisms of RW16 with both zwitterionic and anionic lipid model systems (used as representative models of healthy vs tumor cells) were investigated using a large panoply of biophysical techniques. It was shown that RW16 autoassociates and that its oligomerization state highly influences its membrane interaction. Overall a stronger association and perturbation of anionic membranes was observed, especially in the presence of oligomeric peptide, when compared to zwitterionic ones. This might explain, at least in part, the anti-tumor activity and so the selective interaction with cancer cells whose membranes have been shown to be especially anionic. Hydrophobic contacts between the peptide and lipids were also shown to play an important role in the interaction. That probably results from the tryptophan insertion into the fatty acid lipid area following a peptide flip after the first electrostatic recognition. A model is presented that reflects the ensemble of results. [Copyright &y& Elsevier]

Abstract: Transgenesis is a process that introduces exogenous nucleic acids into the genome of an organism to produce desired traits or evaluate function. Improvements of transgenic technologies are always important pursuit in the last decades. Recently, cell-penetrating peptides (CPPs) were studied as shuttles that can internalize into cells directly and serve as carriers to deliver different cargoes into cells. In the present study, we evaluate whether arginine-rich CPPs can be used for gene delivery into human cells in a noncovalent fashion. We demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) are able to transport plasmid DNA into human A549 cells. For the functional gene assay, the CPP-delivered plasmid DNA containing the enhanced green fluorescent protein (EGFP) coding sequence could be actively expressed in cells. The treatment of calcium chloride did not facilitate the CPP-mediated transfection efficiency, but enhance the gene expression intensity. Mechanistic studies further revealed that HR9/DNA complexes mediate the direct membrane translocation pathway for gene delivery. Our results suggest that arginine-rich CPPs, especially HR9, appear to be a high efficient and promising tool for gene transfer. [Copyright &y& Elsevier]

Abstract: Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84–93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis. [Copyright &y& Elsevier]

Abstract: Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9>PR9>SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans. [Copyright &y& Elsevier]

Abstract: The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called “transposoduction”, which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics. [Copyright &y& Elsevier]

Abstract: Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption. [Copyright &y& Elsevier]

Abstract: We investigated the relationship between intermolecular binding and the ability of novel cell-penetrating peptides (CPPs) to enhance the nasal absorption of therapeutic peptides and proteins. The absorption-enhancing effect of a novel l-penetratin analogue, ‘shuffle (R,K fix) 2’ coadministered with different biotherapeutic peptides was evaluated after nasal administration in rats. Shuffle (R,K fix) 2 significantly increased the nasal absorption of insulin, glucagon-like-peptide-1 (GLP-1) and exendin-4, compared with the absorption seen with l-penetratin. Intermolecular binding was analyzed by surface plasmon resonance (SPR)-based binding assay. The binding characteristics implied that the higher the amount of CPP bound, the greater the nasal drug absorption. In addition, the calculated binding ratio between CPP and drug proved a critical aspect in enhancing the absorption of insulin and GLP-1. This difference in the enhancing effect of CPPs on nasal drug absorption is attributed to the degree of binding with the therapeutic macromolecule. [Copyright &y& Elsevier]

Abstract: The goal of this study was to evaluate whether cell-penetrating peptides (CPPs) affect the nasal absorption of insulin. L- or D-forms of penetratin, or the L- or D-forms of octaarginine (L- or D-R8), was used as first time for nasal insulin delivery. Furthermore, the concentration of lactate dehydrogenase (LDH) in nasal lavage fluid was determined and a histopathological study of nasal respiratory epithelium was conducted. CPPs dramatically increased nasal insulin absorption, and it was more pronounced for L- and D-penetratin than L- or D-R8. L-penetratin was the most effective promoter of insulin absorption compared with others CPPs. A dose-dependent relationship of L-penetratin and insulin bioavailability was statically significant. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively. In contrast, increasing the D-penetratin concentration decreased the efficiency of nasal insulin absorption. There was no significant difference in the release of LDH in nasal lavage fluid and the integrity of nasal respiratory epithelium when L-penetratin was present. In conclusion, these data demonstrate that L-penetratin markedly increased the permeability of insulin across the nasal membrane without causing detectable damage to the integrity of cells in the nasal respiratory mucosa. [Copyright &y& Elsevier]

Abstract: Cell-penetrating peptides (CPPs) are a useful tool for delivering therapeutic macromolecules across cell membranes. We previously devised an approach using CPPs without intermolecular cross-linking and showed the efficient delivery of insulin from the intestine to the systemic circulation using a typical CPP, oligoarginine. However, this approach required relatively high doses of the CPP. Therefore, this study aimed to identify CPPs that are more effective for the delivery of insulin and do not induce toxic effects on the intestine. In this study, we examined the effects of various types of CPPs including arginine-rich peptides and amphipathic peptides that aid insulin absorption from rat ileal segments. Among these peptides, coadministration of insulin with R8, penetratin, pVEC, and RRL helix significantly increased ileal insulin absorption compared with insulin administration alone. In the case of R8, the d-form of the peptide had stronger absorption enhancing ability than the l-form. In contrast, the other three peptides exerted a more significant effect when the l-forms were applied, and l-penetratin had the strongest ability to enhance intestinal insulin absorption. Meanwhile, in a physical mixture of CPP and insulin, aggregates formed in the solution when high concentrations of CPPs were present. l-penetratin enhanced insulin absorption even when administered in an aggregated solution. We then showed that aggregates of l-penetratin and insulin were broken down in the presence of intestinal degradation enzymes. Thus, among CPPs used in this study, l-penetratin had the strongest ability to improve insulin intestinal absorption. [Copyright &y& Elsevier]

Abstract: Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson''s disease. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats. Peptides composed of six (R6), eight (R8) and 10 (R10) residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone; however, insulin absorption increased dramatically after coadministration of the d-form of R6 (d-R6) and the l-form of R6 (l-R6) in a dose-dependent manner. The effects on insulin absorption were more pronounced for d-R6 than for l-R6. Among oligoarginines composed of six, eight, or 10 arginine residues, d-R8 showed the strongest enhancing effects on insulin intestinal absorption. In contrast, intestinal absorption of other model hydrophilic macromolecules, interferon-β and fluorescein isothiocyanate-labeled dextran 4400, was not affected by coadministration with oligoarginine. Pretreatment by the effective dose of l-R6 did not induce lactate dehydrogenase leakage or histological damage, suggesting that oligoarginine has no untoward effect on the intestinal mucosa. Our data demonstrate that coadministration of oligoarginine increases intestinal insulin absorption markedly without causing detectable damage in cellular integrity and that the covalent binding between insulin and oligoarginine is not necessary for this effect. We conclude that oligoarginines are likely to become powerful tools for overcoming the low permeability of insulin through the epithelial cell membrane, the major barrier to oral insulin delivery. [Copyright &y& Elsevier]

Abstract: Glial-cell-line-derived neurotrophic factor (GDNF) promotes mesencephalic dopaminergic neuronal survival in several in vitro and in vivo models. As the demise of dopaminergic neurons is the cause for Parkinson's disease (PD) symptoms, GDNF is a promising agent for its treatment. However, this neurotrophin is unable to cross the blood–brain barrier, which has complicated its clinical use. Therefore, ways to deliver GDNF into the central nervous system in an effective manner are needed. The HIV-1-Tat-derived cell-penetrating peptide (CPP) provides a means to deliver fusion proteins into the brain. We generated a fusion protein between the 11 amino acid CPP of Tat and the rat GDNF mature protein to deliver GDNF across the blood–brain barrier. We showed previously that Tat-GDNF enhances the neuroprotective effect of GDNF in in vivo models for nerve trauma and ischemia. Here, we tested its effect in a subchronic scheme of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) application into the mouse as a model for PD to evaluate the effect of Tat-GDNF fusion protein in dopaminergic neuron survival. We showed that the fusion protein did indeed reach the dopaminergic neurons. However, the in vivo application of Tat-GDNF did not provide neuroprotection of dopaminergic neurons, as revealed by immunohistochemistry and counting of the number of tyrosine-hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Possibly, GDNF does protect nigro-striatal projections of those neurons that survive MPTP treatment but does not increase the number of surviving dopaminergic neurons. A concomitant treatment of Tat-GDNF with an anti-apoptotic Tat-fusion protein might be beneficial. [Copyright &y& Elsevier]

Abstract: During the last decade, small peptides (10 to 15 amino acids) derived from the HIV-1 Tat protein and from the drosophila Antennapedia homeodomain have been used to internalize various types of molecules into the cells. The way these peptides enter cells is still under investigation and the object of strong controversy. The main discussions rely on whether these peptides are internalized or not in an energy-independent fashion, and, depending on the situation, whether they follow one pathway instead of another. At present, we find in the literature a very large number of data with, at times, some contradictory results. Indeed the diversity of employed peptide sequences, the cell type used, the attachment or not of a cargo molecule, the chemical nature of this cargo itself, and the followed protocol during the experimental process do not simplify the comparison and hence final conclusions about the mechanism of cell entry. However, one common feature emerges with these cell-penetrating peptides: most of them do not show any cell specificity. Despite their demonstrated efficiency in delivering biologically active molecules in in vitro experiments, their use for a therapeutic application in vivo has been the object of a relatively little number of studies, probably because of the quite important amounts of CPP–cargo that needs to be prepared for an accurate and complete in vivo study, but more likely, because of the massive spreading of the cargo all around the body. However, it appears from recent studies that an increased targeting ability of these CPPs is possible, making the use of CPP mediated delivery compatible with an in vivo therapeutic approach. [Copyright &y& Elsevier]

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb3+-containing liposomes. Upon chelation of Tb3+ by an aromatic carboxylic acid, the fluorescence of Tb3+ increases many fold. The CPP TAT(44–57)-SAM and TAT(37–53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44–57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44–57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44–57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively. [Copyright &y& Elsevier]

The most commonly studied of the cell-penetrating peptides (CPP) is “penetratin” (pAntp), which functions as a carrier (vector), even for large hydrophilic (cargo) molecules. pAntp originates from the third helix of the Antennapedia homeodomain protein. The peptide is known to interact with negatively charged phospholipid vesicles, which leads to induction of secondary structure. In the present study, circular dichroism (CD) spectroscopy has been used to characterize the different secondary structures induced upon interaction with small unilamellar vesicles (SUVs) from mixtures of zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG). The interaction was monitored using an electron paramagnetic resonance (EPR) spin probe attached to the peptide, and the intrinsic fluorophore (tryptophan). We measured the secondary structure as a function of surface charge density, total lipid-to-peptide (L/P) molar ratio, and salt concentration, for completely bound peptide. With vesicles from POPG/POPC in a molar ratio below 30:70, at a high L/P, the peptide adopts a mainly helical conformation. Increasing the charge density, at the same L/P, promotes a higher degree of β-structure. At a fixed charge density, reducing the L/P also results in an α→β structure conversion. Hence, low membrane surface charge density and low pAntp concentration both favor a mainly helical conformation, while high charge density and pAntp concentration promote a dominating β-structure. We conclude that pAntp, when residing at the surface of a membrane, is chameleon-like in terms of its induced structure. [Copyright &y& Elsevier]