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Structures, toxicity and internalization of cell-penetrating peptides
Stockholm University, Faculty of Science, Department of Neurochemistry.
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 [en]
CPP, cell-penetrating peptide, internalization, toxicity, structures
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-38607ISBN: 978-91-7447-058-1 (print)OAI: oai:DiVA.org:su-38607DiVA: diva2:311253
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
List of papers
1. Cell-Penetrating Peptides: A Comparative Membrane Toxicity Study
Open this publication in new window or tab >>Cell-Penetrating Peptides: A Comparative Membrane Toxicity Study
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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.

Keyword
Cell-penetrating peptides; Membrane perturbation; Lactate dehydrogenase leakage; Hemolytic activity
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-24713 (URN)10.1016/j.ab.2005.07.033 (DOI)
Note
Part of urn:nbn:se:su:diva-7391Available from: 2008-03-04 Created: 2008-03-03 Last updated: 2015-04-21Bibliographically approved
2. Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion
Open this publication in new window or tab >>Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion
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2010 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 6, 1119-1128 p.Article in journal (Refereed) Published
Abstract [en]

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

Keyword
Cell-penetrating peptides, Structure, Membrane interactions, Conformation, Versatility
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-39079 (URN)10.1016/j.bbamem.2010.03.005 (DOI)000278321200012 ()
Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2017-12-12Bibliographically approved
3. An improved synthesis of releasable luciferin-CPP conjugates
Open this publication in new window or tab >>An improved synthesis of releasable luciferin-CPP conjugates
2009 (English)In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 50, no 33, 4731-4733 p.Article in journal (Refereed) Published
Abstract [en]

We have improved the synthesis of a previously published luciferin-linker, used in an assay enabling rapid real-time quantification of luciferin–CPP conjugate uptake and cytosolic cargo release. We also present the synthesis of a new luciferin-linker with the same conjugation ability. Both luciferin-linkers are now available via an efficient one-pot procedure.

Keyword
Luciferin, Luciferin-linker, Conjugate, Cell-penetrating peptide, Releasable luciferin
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-32171 (URN)10.1016/j.tetlet.2009.06.038 (DOI)000268345700011 ()
Available from: 2009-12-04 Created: 2009-12-04 Last updated: 2017-12-12Bibliographically approved
4. Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
Open this publication in new window or tab >>Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
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2010 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 9, 1662-1672 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB I, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.

Keyword
ARGININE-RICH PEPTIDES; REAL-TIME ANALYSIS; HIV-1 TAT PROTEIN; PENETRATING PEPTIDE; MAMMALIAN-CELLS; FIREFLY LUCIFERASE; PLASMA-MEMBRANE; GENE-EXPRESSION; DRUG-DELIVERY; NUCLEIC-ACIDS
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-50078 (URN)10.1021/bc100174y (DOI)000281726500011 ()
Note

authorCount :5

Available from: 2011-01-03 Created: 2010-12-21 Last updated: 2017-12-11Bibliographically approved

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