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Cell-Penetrating Peptides: A Comparative Membrane Toxicity Study
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.
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2005 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 1, 55-65 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43–58), pTAT(48–60), pVEC(615–632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43–58), pTAT(48–60), and pVEC(615–632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 μM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.

Place, publisher, year, edition, pages
2005. Vol. 345, no 1, 55-65 p.
Keyword [en]
Cell-penetrating peptides; Membrane perturbation; Lactate dehydrogenase leakage; Hemolytic activity
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-24713DOI: 10.1016/j.ab.2005.07.033OAI: oai:DiVA.org:su-24713DiVA: diva2:198173
Note
Part of urn:nbn:se:su:diva-7391Available from: 2008-03-04 Created: 2008-03-03 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Internalization mechanisms of cell-penetrating peptides: Structure-activity relationships, cellular distribution and membrane toxicity studies
Open this publication in new window or tab >>Internalization mechanisms of cell-penetrating peptides: Structure-activity relationships, cellular distribution and membrane toxicity studies
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transport of substances with pharmaceutical potential over the plasma membrane has been pushed a great step forward with the discovery of short cationic polypeptides, known as cell-penetrating peptides (CPP). Since then, the internalization mechanism and cargo delivery properties of CPPs has been extensively studied. Unfortunately, no consensus over the internalization mechanism has been achieved so far. Much attention has been paid to the delivery of various macromolecules into the cytoplasm and other subcellular compartments with the help of CPPs, while the structural requirements and toxicity of the peptides and their conjugates has not been studied as thoroughly.

The focus of this thesis is on the characterization of internalization mechanism, toxicity and prediction of CPPs as currently most discussed issues in the field of research.

The studies included in the present thesis indicate that for the majority of CPPs the uptake is dependent of endocytic processes. However, these studies do not reveal one major endocytosis pathway, responsible for the uptake of all tested CPPs. Instead it is most likely that different mechanisms are involved in the uptake of different CPPs. Peptides with apparent amphipathic natures are mainly internalized via clathrin-mediated endocytosis while the CPPs with low amphipathic moment utilize macropinocytosis.

Disturbance of plasma membrane by CPPs in different cell lines was also studied. Results show that peptides with higher amphipathic moment have, in general, higher toxicity, both long- and short-term. However, the amphipathic nature of CPPs is not the only criterion for membrane perturbation. Results also demonstrated the difference in membrane perturbation in different cell lines.

Taken together the studies involved in this thesis provide useful information about the internalization mechanisms and cytotoxicity of cell-penetrating peptides.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 62 p.
Keyword
Cell-penetrating peptides, mechanisms, toxicity, structure-activity relationship
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7391 (URN)978-91-7155-600-4 (ISBN)
Public defence
2008-04-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-03-04 Created: 2008-03-03 Last updated: 2012-02-09Bibliographically approved
2. Structures, toxicity and internalization of cell-penetrating peptides
Open this publication in new window or tab >>Structures, toxicity and internalization of cell-penetrating peptides
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular internalization is a highly regulated process controlled by proteins in the plasma membrane. Large and hydrophilic compounds, in particular, face difficulties conquering the plasma membrane barrier in order to gain access to intracellular environment. This puts serious constrains on the drug industry since many drugs are hydrophilic. Several methods aiming at aiding the cellular internalization of otherwise impermeable compounds have therefore been developed. One such class, so-called cell-penetrating peptides (CPPs), emerged around twenty years ago. This group constitutes hundreds of peptides that have shown a remarkable ability in translocating diverse molecules, ranging from small molecules to large proteins, over the cell membrane. The internalization mechanism of CPPs has been questioned ever since the first peptides were discovered. Initially, the consensus in the field was direct translocation but endocytosis has gradually gained ground. The confusion and the disunity within this research field through the years proceeds from divergent results between research groups that hamper comparison of the peptides.

This thesis aims at characterizing several well-established CPPs with comprehensive studies on cellular toxicity, secondary structure and cellular internalization kinetics.

The results demonstrate that CPPs act in general in a low or non-toxic way, but the apparent toxicity is both peptide- and cell line-dependent. Structural studies show that the CPPs have a diverse polymorphic behavior ranging from random coil to structured β-sheet or α-helix, depending on the environment. The ability to change secondary structure could be the key to the internalization property of the CPPs. Internalization kinetic studies of CPP conjugates reveal two sorts of internalization profiles, either fast curves that cease in few minutes or slow curves that peak in tens of minutes. Furthermore, improved synthesis of CPP conjugates is demonstrated.

In conclusion, the studies in this thesis provide useful information about cytotoxicity and structural diversity of CPPs, and emphasize the importance of kinetic measurements over end-point studies in order to give better insights into the internalization mechanisms of CPPs.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholms University, 2010. 54 p.
Keyword
CPP, cell-penetrating peptide, internalization, toxicity, structures
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-38607 (URN)978-91-7447-058-1 (ISBN)
Public defence
2010-06-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.

Available from: 2010-05-11 Created: 2010-04-20 Last updated: 2015-03-11Bibliographically approved

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