Cell-penetrating peptide secures an efficient endosomal escape of an intact cargo upon a brief photo-induction
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Since their discovery, cell-penetrating peptides (CPPs) have provided a novel, efficient, and non-invasive mode of transport for various (bioactive) cargos into cells. Despite the ever-growing number of successful implications of the CPP-mediated delivery, issues concerning their intracellular trafficking, significant targeting to degradative organelles, and limited endosomal escape are still hindering their widespread use. To overcome these obstacles, we have utilized a potent photo-induction technique with a fluorescently labeled protein cargo attached to an efficient CPP, TP10. In this study we have determined some key requirements behind this induced escape (e.g., dependence on peptide-to-cargo ratio, time and cargo), and have semi-quantitatively assessed the characteristics of the endosomes that become leaky upon this treatment. Furthermore, we provide evidence that the photo-released cargo remains intact and functional. Altogether, we can conclude that the photo-induced endosomes are specific large complexes-condensed non-acidic vesicles, where the released cargo remains in its native intact form. The latter was confirmed with tubulin as the cargo, which upon photo-induction was incorporated into microtubules. Because of this, we propose that combining the CPP-mediated delivery with photo-activation technique could provide a simple method for overcoming major limitations faced today and serve as a basis for enhanced delivery efficiency and a subsequent elevated cellular response of different bioactive cargo molecules.
KeywordsCPP Protein transduction domain PTD Photo-induction Effective delivery Endosomal release Degradation Stability
Transportan 10, a shortened analogue of transportan (TP)
N-terminally biotinylated TP10
The study was supported by grants from the Estonian Science Foundation (ESF 7058 and 8705) and the Estonian Ministry of Education and Research (0182691s05, 0180019s11, and 0180027s08). Ü. L. was also supported by the Swedish Research Council (VR-NT); by the Center for Biomembrane Research, Stockholm; by the Knut and Alice Wallenberg’s Foundation; by the EU through the European Regional Development Fund through the Center of Excellence in Chemical Biology, Estonia. H. R. was supported by Olev and Talvi Maimets Stipend (Foundation of the University of Tartu) and Artur Lind Stipend (Estonian Genome Foundation). The authors declare no conflicts of interest.
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