| 1. |
- Arukuusk, Piret, et al.
(författare)
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New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids
- 2013
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1828:5, s. 1365-1373
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Tidskriftsartikel (refereegranskat)abstract
- Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.
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| 2. |
- Copolovici, Dana Maria, et al.
(författare)
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Cell-Penetrating Peptides : Design, Synthesis, and Applications
- 2014
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 8:3, s. 1972-1994
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Forskningsöversikt (refereegranskat)abstract
- The intrinsic property of cell-penetrating peptides (CPPs) to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be potential components of future drugs and disease diagnostic agents. These versatile peptides are simple to synthesize, functionalize, and characterize yet are able to deliver covalently or noncovalently conjugated bioactive cargos (from small chemical drugs to large plasmid DNA) inside cells, primarily via endocytosis, in order to obtain high levels of gene expression, gene silencing, or tumor targeting. Typically, CPPs are often passive and nonselective yet must be functionalized or chemically modified to create effective delivery vectors that succeed in targeting specific cells or tissues. Furthermore, the design of clinically effective systemic delivery systems requires the same amount of attention to detail in both design of the delivered cargo and the cell-penetrating peptide used to deliver it.
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| 3. |
- Eriste, Elo, et al.
(författare)
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Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2
- 2013
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 24:3, s. 305-313
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Tidskriftsartikel (refereegranskat)abstract
- Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using in vitro phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where in vitro phage display has yielded an efficient, in vivo working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems.
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| 4. |
- Jones, Sarah, et al.
(författare)
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Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa : evaluation of efficient delivery vectors that do not compromise human sperm motility
- 2013
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Ingår i: Human Reproduction. - : Oxford University Press (OUP). - 0268-1161 .- 1460-2350. ; 28:7, s. 1874-1889
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Tidskriftsartikel (refereegranskat)abstract
- Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples. CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. Chemically heterogeneous CPPs readily translocated into sperm to accumulate within discrete intracellular compartments. Mitoparan (INLKKLAKL(Aib)KKIL), for example, specifically accumulated within the mitochondria located in the sperm midpiece. The unique plasma membrane composition of sperm is a critical factor that directly influences the uptake efficacy of structurally diverse CPPs. No correlations in efficacies were observed when comparing CPP uptake into sperm with either uptake into fibroblasts or direct translocation across a phosphatidylcholine membrane. These comparative investigations identified C105Y (CSIPPEVKFNKPFVYLI) as a most efficient pharmacokinetic modifier for general applications in sperm biology. Significantly, CPP uptake induced no detrimental influence upon either bovine sperm viability or the motility of human sperm. As a consequence of the lack of endocytotic machinery, the CPP-mediated delivery of much larger protein complexes into sperm is relatively inefficient when compared with the similar process in fibroblasts. It is possible that some CPPs could directly influence aspects of sperm biology and physiology that were not analysed in this study. CPP technologies have significant potential to deliver selected bioactive moieties and so could modulate the biology and physiology of human sperm biology both prior- and post-fertilization. We are pleased to acknowledge financial support from the following sources: the Wellcome Trust, TENOVUS (Scotland), University of Dundee, Medical Research Council, NHS Tayside and Scottish Enterprise and the Research Institute in Healthcare Science, University of Wolverhampton. No conflicts of interest are reported by the authors.
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| 5. |
- Lehto, Taavi, et al.
(författare)
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A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
- 2011
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 19:8, s. 1457-1467
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Tidskriftsartikel (refereegranskat)abstract
- Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.
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| 6. |
- Lehto, Taavi, et al.
(författare)
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Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy
- 2010
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 141:1, s. 42-51
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000. We show that stearyl-(RxR)4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR)4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)4 promotes dose-dependent splice correction in parity with (RxR)4-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)4 an intriguing vector for future in vivo experiments.
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| 7. |
- Oskolkov, Nikita, et al.
(författare)
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NickFects, Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides
- 2011
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Ingår i: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 17:2, s. 147-157
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Tidskriftsartikel (refereegranskat)abstract
- Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides. These carriers, obtained by replacement of Ile8 by threonine in stearyl-TP10 and by modifying of tyrosine and/or threonine, respectively, by phosphorylation formed 300-500 nm in size peptide: oligonucleotide nanocomplexes with negative surface charges. The highest splice-correcting effect was obtained when phosphorotiate 2'-O-methyl oligonucleotides were transduced into cells by NickFect1 (NF1) or NickFect2 (NF2). In addition, we also show how a small modification (one or two negative charges) in peptide sequence can affect its ability to deliver ONs into cells and increase their potency in the splicing redirection assay. Our studies demonstrate that NF1 and NF2 have higher transfection efficacy for oligonucleotides as compared to the most commonly used transfection agent Lipofectamine (TM) 2000 and lead to higher biological response in cells.
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| 8. |
- Suhorutsenko, Julia, et al.
(författare)
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Cell-Penetrating Peptides, PepFects, Show No Evidence of Toxicity and Immunogenicity In Vitro and In Vivo
- 2011
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 22:11, s. 2255-2262
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1 beta, IL-18, and TNF-alpha cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 mu M and 5 mu M, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.
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| 9. |
- Suhorutsenko, Julia, et al.
(författare)
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Human protein 53 derived cell penetrating peptides
- 2012
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 18:4, s. 291-297
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Tidskriftsartikel (refereegranskat)abstract
- Tumor suppressor protein 53 plays an important role in the initiation of cell cycle arrest and apoptosis. Being highly mutated in several different cancer types, p53 is a good target for anticancer therapeutics. It has been shown that a peptide derived from the C-terminus of p53 activates specific DNA-binding of endogenous mutated p53, restoring its original activity. Detection of short cell-penetrating peptide sequences using quantitative structure-activity relationship algorithm gives new opportunities for developing novel peptide-based platforms for modulation of biological activity inside the cell. Here we present novel human protein 53 C-terminal domain-derived peptides, Peptide4 and Peptide5 that were designed using cell-penetrating peptide prediction algorithm and synthesised by Fmoc chemistry. Peptide4 and Peptide5 showed to be capable for translocation inside the breast cancer cells. Subsequent introduction of stearic acid moiety in the backbone of these peptides at N-terminal or lysine 3-orthogonal positions enhanced their cell-penetrating ability. Moreover Peptide4 and Peptide5 showed certain cytotoxic activity and were able to induce apoptosis in MDA-MB-231 cell line in the absence of serum. We suggest that human protein 53 C-terminal domain-derived cell-penetrating peptides Peptide4 and Peptide5 have promising perspectives for the future anticancer applications.
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