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Differential Endosomal Pathways for Radically Modified Peptide Vectors
Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-8813-1096
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2013 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 10, 1721-1732 p.Article in journal (Refereed) Published
Abstract [en]

In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.

Place, publisher, year, edition, pages
2013. Vol. 24, no 10, 1721-1732 p.
National Category
Biochemistry and Molecular Biology Chemical Sciences
URN: urn:nbn:se:su:diva-96658DOI: 10.1021/bc4002757ISI: 000326125500009OAI: diva2:667484


Available from: 2013-11-26 Created: 2013-11-25 Last updated: 2015-04-22Bibliographically approved
In thesis
1. Oligonucleotide Complexes with Cell-Penetrating Peptides: Structure, Binding, Translocation and Flux in Lipid Membranes
Open this publication in new window or tab >>Oligonucleotide Complexes with Cell-Penetrating Peptides: Structure, Binding, Translocation and Flux in Lipid Membranes
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles.

The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems.

We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014. 32 p.
Cell-penetrating peptide, Large unilamellar vesicle, Membrane perturbation, Endosomal escape
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
urn:nbn:se:su:diva-109299 (URN)978-91-7649-029-7 (ISBN)
2014-11-17, C 458 (Heilbronnsalen), Svante Arrhenius väg 16 B, Stockholm, 16:55 (English)
Available from: 2014-11-17 Created: 2014-11-17 Last updated: 2015-03-17Bibliographically approved

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Eriksson, N. K. JonasVasconcelos, LuisLangel, Ülo
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