| 1. |
- Copolovici, Dana Maria, et al.
(author)
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Cell-Penetrating Peptides : Design, Synthesis, and Applications
- 2014
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 8:3, s. 1972-1994
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Research review (peer-reviewed)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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| 2. |
- Eriste, Elo, et al.
(author)
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Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2
- 2013
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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 24:3, s. 305-313
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Journal article (peer-reviewed)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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| 3. |
- Eriste, Elo
(author)
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Purification, structure and function of bioactive peptides
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Peptides are vitally important molecules and many evoke cellular responses. The completion of several genome sequencing projects has revealed a number of new genes. However, as functional peptides often contain posttranslational modifications and/or occur at various lengths, it is of great importance to detect, purify and characterize novel bioactive peptides. To achieve these goals, new methods for peptide detection, isolation and functional characterization have to be developed. In the present thesis, a broad range of different techniques for detection, purification, structural and functional characterization of new peptides, the production of recombinant peptides, their reconstitution with metals and functional characterization was utilized. A detection method was developed to screen for receptor ligands from tissue extracts using Chinese hamster ovary (CHO) cells with a Cytosensor. As a proof-of-concept, an active component in tissue extracts was purified by a seven-step protocol. The structure was determined by mass spectrometry (MS), which identified it as insulin-like growth factor-I (IGF-I). A novel ligand for an orphan G-protein coupled receptor, GPCR135, was detected in porcine brain extracts by guanosine 5`-O-(3-thiotriphosphate) (GTPγS) incorporation assay. The ligand was purified by a six-step procedure. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry analyses demonstrated that the ligand is relaxin 3, which is composed of two peptide chains linked together with disulfide bridges similarly to insulin-relaxin family of peptides. Pharmacological experiments showed that relaxin-3 is the only ligand that activates GPCR135. An elongated form of peptide histidine isoleucine amide (PHI), PHI-42, was detected in porcine intestinal extracts by cAMP assays on IMR-32 neuroblastoma cells. PHI-42 was purified from the mixture by a four-step procedure. The structure was determined by MALDI MS and Edman degradation, which showed that the 12-residue sequence of porcine PHI-42 is unique and links together PHI-27 and vasoactive intestinal polypeptide (VIP) in the precursor. A novel posttranslationally modified form of neurotensin (NT) was identified from porcine intestine and purified by a five-step procedure. Linear ion trap-Fourier transform, MALDI, and ESI quadropole time-of-flight (Q-TOF) mass spectrometries identified the posttranslational modification as an Arg-residue coupled to the gamma-carboxyl group of Glu-4 by an isopeptide bond. In addition, NT(2-13) and NT(3-13) together with a fragment (22-38) of dopamine- and adenosine 3 ,5 -monophosphate-regulated phosphoprotein (DARPP-32) were also detected and studied by MALDI MS and Edman degradation. All the NT forms were active in intracellular Ca2+ release assay. Human brain-specific metallothionein-3 (MT-3) was produced by recombinant technology and studied by ESI MS. The protein was purified as apo-MT-3 by a new three-step procedure. Titration of MT-3 with Zn2+ and Cd2+ ions and the stability of metal complexes were followed by ESI MS and the results were compared with data for common MT-s. We demonstrated that MT-3, in contrast to common MTs, binds divalent metals in a non-cooperative manner and at higher capacity than common MTs. The combination of bioassays, high performance liquid chromatography, and mass spectrometry has enabled us to detect, purify and characterize novel peptides from biological sources, yielding new knowledge about their localization, structure, processing and functions.
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| 4. |
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| 5. |
- Lehto, Taavi, et al.
(author)
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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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In: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 141:1, s. 42-51
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Journal article (peer-reviewed)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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| 6. |
- Pärn, Kalle, et al.
(author)
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The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides
- 2015
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In: Cell-Penetrating Peptides. - New York, NY : Springer-Verlag New York. - 9781493928057 - 9781493928064 ; , s. 223-245
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Book chapter (peer-reviewed)abstract
- Over the past two decades, cell-penetrating peptides (CPPs) have become increasingly popular both in research and in application. There have been numerous studies on the physiochemical characteristics and behavior of CPPs in various environments; likewise, the mechanisms of entry and delivery capabilities of these peptides have also been extensively researched. Besides the fundamental issues, there is an enormous interest in the delivery capabilities of the peptides as the family of CPPs is a promising and mostly non-toxic delivery vector candidate for numerous medical applications such as gene silencing, transgene delivery, and splice correction. Lately, however, there has been an emerging field of study besides the high-profile gene therapy applications-the use of peptides and CPPs to combat various infections caused by harmful bacteria, fungi, and viruses.In this chapter, we aim to provide a short overview of the history and properties of CPPs which is followed by more thorough descriptions of antimicrobial and antiviral peptides. To achieve this, we analyze the origin of such peptides, give an overview of the mechanisms of action and discuss the various practical applications which are ongoing or have been suggested based on research.
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| 7. |
- Suhorutsenko, Julia, et al.
(author)
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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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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 22:11, s. 2255-2262
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Journal article (peer-reviewed)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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| 8. |
- Suhorutsenko, Julia, et al.
(author)
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Human protein 53 derived cell penetrating peptides
- 2012
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In: 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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Journal article (peer-reviewed)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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