Activation of cell-penetrating peptide fragments by disulfide formation
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ORIGINAL ARTICLE
Activation of cell‑penetrating peptide fragments by disulfide formation Raheleh Tooyserkani1 · Wojciech Lipiński1 · Bob Willemsen1 · Dennis W. P. M. Löwik1 Received: 21 April 2020 / Accepted: 26 July 2020 © The Author(s) 2020
Abstract Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery. Keywords Cell-penetrating peptide · Cellular uptake · Disulfide conjugation · Tat · Pep-3 · Penetratin
Introduction In the search for new, efficient delivery methods of therapeutic molecules and particles into cells, cell-penetrating peptides (CPPs) emerged 30 years ago as extraordinary vector molecules (Copolovici et al. 2014). They can facilitate transmembrane transport through a receptor-independent pathway and without inducing toxicity. However, despite a great number of both natural and synthetic CPPs that have been discovered, varying in their chemical structure and exhibiting different properties, the common use of CPPs as delivery vectors is hampered due to a number of practical difficulties (Feni and Neundorf 2017; Shi et al. 2014). Firstly, because the mechanism of cellular uptake remains elusive in many cases, and as it seems to be significantly dependent on the type of cargo, type of cells and experimental conditions, it is Handling editor: F. Albericio. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00726-020-02880-x) contains supplementary material, which is available to authorized users. * Dennis W. P. M. Löwik [email protected] 1
Radboud University Nijmegen, Institute for Molecules and Materials, Bio-Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
very difficult to develop an all-embracing delivery solution based on CPPs (Madani et al. 2011). Secondly, and probably even more important, CPPs are taken up non-selectively in a great variety of cells. Hence, several strategies have been developed to improve the selectivity of CPP-based delivery platforms. For example, CPPs or CPP-based delivery platforms can be coupled to targeting ligands (e.g., hyaluronic acid, (Yamada et al. 2015) folic acid (Li et al. 2016), transferrin (Sharma et al. 2016), bombesin (Nallely et al. 2013), RGD or NGR sequence (Xie et al. 2016)) or ca
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