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Peptide degradation is a critical determinant for cell-penetrating peptide uptake
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-8947-6643
2007 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1768, no 7, 1769-1776 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

Place, publisher, year, edition, pages
2007. Vol. 1768, no 7, 1769-1776 p.
Keyword [en]
cell-penetrating peptide, protein transduction domains, uptake, degradation, endocytosis inhibitor
National Category
Biochemistry and Molecular Biology Biophysics
URN: urn:nbn:se:su:diva-25531DOI: 10.1016/j.bbamem.2007.03.029ISI: 000247716200009OAI: diva2:199912
Available from: 2008-10-23 Created: 2008-10-07 Last updated: 2015-03-09Bibliographically approved
In thesis
1. Design and evaluation of drug delivery vehicles
Open this publication in new window or tab >>Design and evaluation of drug delivery vehicles
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 77 p.
National Category
Research subject
Neurochemistry and Neurotoxicology
urn:nbn:se:su:diva-8256 (URN)978-91-7155-754-4 (ISBN)
Public defence
2008-11-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Available from: 2008-10-23 Created: 2008-10-07Bibliographically approved

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Hällbrink, Mattias
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ReferencesLink to record
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