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Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
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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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.

Place, publisher, year, edition, pages
2010. Vol. 21, no 9, 1662-1672 p.
Keyword [en]
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: urn:nbn:se:su:diva-50078DOI: 10.1021/bc100174yISI: 000281726500011OAI: oai:DiVA.org:su-50078DiVA: diva2:382680
Note

authorCount :5

Available from: 2011-01-03 Created: 2010-12-21 Last updated: 2017-12-11Bibliographically approved
In thesis
1. 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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