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Ulo Langel - One of the best experts on this subject based on the ideXlab platform.
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pepfect15 a novel endosomolytic cell penetrating Peptide for oligonucleotide delivery via scavenger receptors
International Journal of Pharmaceutics, 2013Co-Authors: Staffan Lindberg, Ulo Langel, Henrik Helmfors, Oana Tudoran, Diogo Mosqueira, Daniel Gyllborg, Andres MunozalarconAbstract:Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-Penetrating Peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel Cell-Penetrating Peptide, PepFect15, comprising the previously reported PepFect14 Peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes Peptide-mediated anti-microRNA delivery. The Peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.
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intracellular delivery of short interfering rna in rat organ of corti using a cell penetrating Peptide pepfect6
Molecular therapy. Nucleic acids, 2012Co-Authors: Suvarna Dashwagh, Ulo Langel, Staffan Lindberg, Stefan Jacob, Anders Fridberger, Mats UlfendahlAbstract:RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel Cell-Penetrating Peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA–treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.
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design of a tumor homing cell penetrating Peptide
Bioconjugate Chemistry, 2008Co-Authors: Helena Myrberg, Maarja Mäe, Lianglin Zhang, Ulo LangelAbstract:Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic Peptide cCPGPEGAGC (PEGA) is a homing Peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA Peptide does not cross the plasma membrane per se; however, when attached to the Cell-Penetrating Peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA-pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA-pVEC chimeric Peptide. These data demonstrate that combining a homing sequence with a Cell-Penetrating sequence yields a Peptide that combines the d...
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characterization of a novel cytotoxic cell penetrating Peptide derived from p14arf protein
Molecular Therapy, 2008Co-Authors: H Johansson, Maarja Mäe, Samir Elandaloussi, Tina Holm, Jaak Janes, Toivo Maimets, Ulo LangelAbstract:The tumor suppressor p14ARF is widely deregulated in many types of cancers and is believed to function as a failsafe mechanism, inhibiting proliferation and inducing apoptosis as cellular response to a high oncogene load. We have found that a 22-amino-acid-long Peptide derived from the N-terminal part of p14ARF, denoted ARF(1–22), which has previously been shown to mimic the function of p14ARF, has Cell-Penetrating properties. This Peptide is internalized to the same extent as the Cell-Penetrating Peptide (CPP) TP10 and dose-dependently decreases proliferation in MCF-7 and MDA MB 231 cells. Uptake of the ARF(1–22) Peptide is associated with low membrane disturbance, measured by deoxyglucose and lactate dehydrogenase (LDH) leakage, as compared to its scrambled Peptide. Also, flow cytometric analysis of annexin V/propidium iodide (PI) binding and Hoechst staining of nuclei suggest that ARF(1–22) induces apoptosis, whereas scrambled or inverted Peptide sequences have no effect. The ARF(1–22) Peptide mainly translocates cells through endocytosis, and is found intact inside cells for at least 3 hours. To our knowledge, this is the first time a CPP having pro-apoptopic activity has been designed from a protein.
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mechanism of the cell penetrating Peptide transportan 10 permeation of lipid bilayers
Biophysical Journal, 2007Co-Authors: Lindsay E Yandek, Ulo Langel, Antje Pokorny, Anders Floren, Kristina Knoelke, Paulo F AlmeidaAbstract:The mechanism of the interaction between the Cell-Penetrating Peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of Peptide association with vesicles and Peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows Peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the Peptide mass imbalance is dissipated. In that transient state tp10 “catalyzes” dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between Peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of Cell-Penetrating and antimicrobial Peptides.
Quyen T Nguyen - One of the best experts on this subject based on the ideXlab platform.
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surgical molecular navigation with ratiometric activatable cell penetrating Peptide for intraoperative identification and resection of small salivary gland cancers
Head and Neck-journal for The Sciences and Specialties of The Head and Neck, 2016Co-Authors: Timon Hussain, Elamprakash N Savariar, Julio A Diazperez, Karen Messer, Roger Y Tsien, Quyen T NguyenAbstract:Background We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable Cell-Penetrating Peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model. Methods Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time, and tumor-free survival were compared. Results RACPP guidance aided tumor detection (positive histology in 90% [27/30] vs 48% [15/31] for WL; p 90% detection sensitivity and specificity. Operating time was reduced by 54% (p < .001), and tumor-free survival was increased with RACPP guidance (p = .025). Conclusion RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications. © 2015 Wiley Periodicals, Inc. Head Neck 38: 715–723, 2016
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surgical molecular navigation with ratiometric activatable cell penetrating Peptide for intraoperative identification and resection of small salivary gland cancers
Head and Neck-journal for The Sciences and Specialties of The Head and Neck, 2016Co-Authors: Timon Hussain, Elamprakash N Savariar, Julio A Diazperez, Karen Messer, Roger Y Tsien, Quyen T NguyenAbstract:Author(s): Hussain, Timon; Savariar, Elamprakash N; Diaz-Perez, Julio A; Messer, Karen; Pu, Minya; Tsien, Roger Y; Nguyen, Quyen T | Abstract: BackgroundWe evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable Cell-Penetrating Peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model.MethodsSurgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time, and tumor-free survival were compared.ResultsRACPP guidance aided tumor detection (positive histology in 90% [27/30] vs 48% [15/31] for WL; p l .001). An approximate 25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded g90% detection sensitivity and specificity. Operating time was reduced by 54% (p l .001), and tumor-free survival was increased with RACPP guidance (p = .025).ConclusionRACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications.
Victor C Yang - One of the best experts on this subject based on the ideXlab platform.
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enzyme triggered cell penetrating Peptide mediated delivery of anti tumor agents
Journal of Controlled Release, 2016Co-Authors: Lu Sun, Victor C Yang, Ergang Liu, Sunhui Chen, Qiuling Liang, Meong Cheol ShinAbstract:Conventional chemotherapy has little or no specificity for cancer cells, normally resulting in low drug accumulation at the tumor region (inefficacy) and drug-induced severe side effects (toxicity). Nowadays, new strategies have been developed to improve both the targeting ability and cellular drug uptake using active targeting ligands and drug internalization agents, which could recognize and interact with specific receptors overexpressed on tumor cells and then trigger a drug internalization process by transporting the cargos into cells. Among those strategies, enzyme-triggered cell penetrating Peptide (CPP)-mediated systems seem to be a feasible approach. The expression level of specific enzymes like proteases, esterases or glycosidases is often higher in tumor cells than in normal tissues, and such concentration gradients can be exploited as a tool for targeted cancer therapy. CPPs are known to be effective in promoting membrane transportation of the drug cargos, rendering a deeper tumor permeation that could further enhance the therapeutic efficacy of the delivered drug. An enzyme-triggered, CPP-mediated system would combine these advantages to yield a system with the enhanced tumor targeting ability and internalization efficiency and so far many systems have been successfully exploited and applied to cancer therapy. In this review, typical enzymes applied in cancer theranostic systems were firstly reviewed, followed by analyzing pros and cons of cell penetrating Peptides. Most importantly, different types of applications of enzyme-triggered CPP-mediated systems in tumor imaging were illustrated. Finally, the drug loaded applications, i.e. enzyme-triggered CPP-mediated systems in drug delivery were reviewed.
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curb challenges of the trojan horse approach smart strategies in achieving effective yet safe cell penetrating Peptide based drug delivery
Advanced Drug Delivery Reviews, 2013Co-Authors: Yongzhuo Huang, Huiyuan Wang, Yifan Jiang, Meong Cheol Shin, Jianxin Wang, Youngro Byun, Huining He, Yanqin Liang, Victor C YangAbstract: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.
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the systemic delivery of sirnas by a cell penetrating Peptide low molecular weight protamine
Biomaterials, 2010Co-Authors: Young Suk Choi, Victor C Yang, Jue Yeon Lee, Jin Sook Suh, Young Min Kwon, Seung Jin Lee, J K Chung, Dong Soo Lee, Chong Pyoung Chung, Yoon Jeong ParkAbstract:Small interfering RNAs (siRNAs), used for specific down-regulation of targeted genes, have garnered considerable interest as an attractive new class of drugs for broad clinical applications. The polyanionic charges carried by these siRNAs, however, restrain cellular uptake and consequently limit effects on gene regulation. Herein the authors describe a Peptide/siRNA complex containing the cell penetrating Peptide derived from natural protamine, termed low molecular weight protamine (LMWP), for the treatment of cancer. Fluorescently-tagged siRNAs were localized with the Peptide in the cytoplasm shortly after incubation of LMWP/siRNA complex with carcinoma cells. The increased cell uptake of siRNA that was achieved using the LMWP resulted in significant down-regulation of model protein luciferase as well as therapeutic cancer target, vascular endothelial growth factor (VEGF) expression. In vivo studies with tumor-bearing mice further demonstrated that the Peptide could carry and localize siRNA inside tumors and inhibit the expression of VEGF through systemic application of the Peptide complex, thereby suppressing tumor growth. In addition, no detectable increase in the serum level of inflammatory cytokines including interferon (IFN)-α and interleukin (IL)-12 was observed under the LMWP/siRNA complex treatment, indicating systemic delivery of LMWP/siRNA did not exert measurable immunostimulatory effect. The LMWP-based systemic delivery method could be a reliable and safe approach to maximize effectiveness of therapeutic siRNA for treatment of cancer and other diseases.
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insulin cell penetrating Peptide hybrids with improved intestinal absorption efficiency
Biochemical and Biophysical Research Communications, 2005Co-Authors: Jun F Liang, Victor C YangAbstract:Abstract Cell-Penetrating Peptide (CPP) was linked to insulin to form insulin–CPP hybrids. The intestinal absorption efficiency of CPP hybridized insulin was 6–8 times increased compared to normal insulin as tested on Caco-2 cell monolayer, a widely used in vitro model for intestinal absorption. Insulin–CPP hybrid transportation seemed to be through an active and transcytosis-like mechanism. Importantly, insulin in hybrids kept intact after they passed through the Caco-2 cell monolayer. This study provides a new clue for oral insulin development.
Jesus Ayala-sanmartin - One of the best experts on this subject based on the ideXlab platform.
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Membrane re-arrangements and rippled phase stabilisation by the cell penetrating Peptide penetratin
Biochimica et Biophysica Acta:Biomembranes, 2016Co-Authors: Claudia Almeida, Antonin Lamaziere, Angélique Filleau, Yohann Corvis, Philippe Espeau, Jesus Ayala-sanmartinAbstract:Cell penetrating Peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20 years ago, the mechanisms of Peptide membrane crossing are still controversial. The different suggested penetration mechanisms reflect the high sequence and structural diversity of cell penetrating Peptides. The fundamental step for Peptide penetration into the cytosol is the crossing of the membrane lipid barrier at the level of the plasma membrane or the endosomes. Therefore, the study of the Peptide-lipid interaction is the key for Peptide penetration mechanisms understanding. In order to study the changes in lipid organisation induced by the cell penetrating Peptide penetratin, several experiments by three different physicochemical approaches were performed. X-ray diffraction data shows that penetratin is able to induce membrane phase separation and lipid rearrangements observed by inter-lipid distances. These changes are accompanied by a temperature stable behaviour of some of the induced membrane domains. The membrane environment fluorescent probe laurdan showed that, in DMPC and DMPC/DMPG membranes, the Peptide induces de-packing of lipids. Calorimetric analyses show that penetratin favours the gel phase to gel-like rippled phase transition. Overall, the data suggest both, that the rippled phase is a heterogeneous structure formed by gel-like and fluid-like coexisting components, and that the penetratin-induced membrane heterogeneity could be important for membrane destabilisation during cell penetration.
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Metabolic energy-independent mechanism of internalization for the cell penetrating Peptide penetratin.
BBA - Biochimica et Biophysica Acta, 2012Co-Authors: Ofelia Maniti, Germain Trugnan, Antonin Lamaziere, Elise Blanchard, Jesus Ayala-sanmartinAbstract:Cellular uptake of vector Peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating Peptide penetratin. The results show that cell penetrating Peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of Peptides to induce their internalization.
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Distinct behaviour of the homeodomain derived cell penetrating Peptide penetratin in interaction with different phospholipids.
PLoS ONE, 2010Co-Authors: Ofelia Maniti, Germain Trugnan, Isabel Alves, Jesus Ayala-sanmartinAbstract:BACKGROUND: Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating Peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β-sheet contributions. CONCLUSIONS/SIGNIFICANCE: These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different Peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.
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Tubular structures in heterogeneous membranes induced by the cell penetrating Peptide penetratin.
Communicative & integrative biology, 2009Co-Authors: Antonin Lamaziere, Germain Trugnan, Gérard Chassaing, Jesus Ayala-sanmartinAbstract:The delivery of active molecules into cells requires the efficient translocation of the plasma membrane barrier. Penetratin is a promising cell penetrating Peptide is which crosses the cell membrane by a receptor and metabolic energy-independent mechanism. In previous work, we have shown that basic Peptides induce membrane invaginations (i.e., tubes formation by induction of negative curvature of membranes) suggesting a new mechanism for cellular uptake of cell penetrating Peptides: “physical endocytosis”. These effects on membrane curvature are favored in pure liquid disordered but not in pure liquid ordered (raft-like) membrane domains. Herein, we present experiments in heterogeneous membranes composed of mixed domains. The results show that Penetratin is able to induce invaginations in membranes in which liquid ordered and liquid disordered membranes coexist. We suggest that Penetratin is able to recruit specific lipids locally forming fluid membrane patches dispersed inside a liquid ordered membrane z...
Jeanchristophe Leroux - One of the best experts on this subject based on the ideXlab platform.
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activatable cell penetrating Peptide Peptide nucleic acid conjugate via reduction of azobenzene peg chains
Journal of the American Chemical Society, 2014Co-Authors: Soo Hyeon Lee, Elena Moroz, Bastien Castagner, Jeanchristophe LerouxAbstract:The use of stimuli-responsive bioactive molecules is an attractive strategy to circumvent selectivity issues in vivo. Here, we report an activatable cell penetrating Peptide (CPP) strategy ultimately aimed at delivering nucleic acid drugs to the colon mucosa using bacterial azoreductase as the local reconversion trigger. Through screening of a panel of CPPs, we identified a sequence (M918) capable of carrying a nucleic acid analogue payload. A modified M918 Peptide conjugated to a Peptide nucleic acid (PNA) was shown to silence luciferase in colon adenocarcinoma cells (HT-29-luc). Reversible functionalization of the conjugate’s lysine residues via an azobenzene self-immolative linkage abolished transfection activity, and the free CPP-PNA was recovered after reduction of the azobenzene bond. This activatable CPP conjugate platform could find applications in the selective delivery of nucleic acid drugs to the colon mucosa, opening therapeutic avenues in colon diseases.