Effect of small molecule signaling in PepFect14 transfection

PLoS One. 2020 Jan 30;15(1):e0228189. doi: 10.1371/journal.pone.0228189. eCollection 2020.


Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. Previous studies have shown that the use of small molecule drugs could be an efficient method to increase the efficacy of delivery of oligonucleotides by cell-penetrating peptides either as targeting agents that can be used in formulation with the cell-penetrating peptide and its cargo or as cell signaling modulators that facilitates the cellular uptake of the treatment. This study presents two aims. The first aim is the identification of small molecule drugs that would induce a synergic effect on the transfection of splice correcting oligonucleotides assisted by PepFect14. The second aim is to identify the mechanisms behind the effect of small molecule drugs modulation of cell-penetrating peptide assisted transfection of oligonucleotides. Through an optimized, high-throughput luciferase assay for short oligonucleotide delivery using cell-penetrating peptides, and the simultaneous addition of a small molecule drug library, we show that three small molecule drugs (MPEP, VU0357121 and Ciproxifan) induced an increase in the transfection efficacy of PepFect14 in complex with a short single-stranded oligonucleotide in HeLa pLuc705 cells. These three drugs are described in the literature to be highly specific for their respective target receptors. However, none of those receptors are expressed in our cell line, indicating a yet non-described pathway of action for these small molecules. We show that the indicated small molecules, without interfering with the particles formed by PepFect14 and the oligonucleotide, interfere via still unidentified interactions in cell signaling, leading to an up-regulation of endocytosis and a higher efficacy in the delivery of short splice correcting oligonucleotides in complex with PepFect14.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzamides / metabolism
  • Cell-Penetrating Peptides / metabolism*
  • Drug Delivery Systems*
  • Endocytosis
  • HeLa Cells
  • High-Throughput Screening Assays
  • Humans
  • Imidazoles / metabolism
  • Lipopeptides / metabolism*
  • Nanoparticles / metabolism
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism*
  • Peptides / metabolism
  • Pyridines / metabolism
  • Receptors, Cell Surface / metabolism
  • Signal Transduction*
  • Transfection*


  • Benzamides
  • Cell-Penetrating Peptides
  • Imidazoles
  • Lipopeptides
  • Oligonucleotides
  • PepFect14 peptide
  • Peptides
  • Pyridines
  • Receptors, Cell Surface
  • VU0357121
  • ciproxifan
  • 6-methyl-2-(phenylethynyl)pyridine

Grant support

This work was supported by the Swedish Research Council (VR 521-2011-2461 to ÜL and VR 2017-03691 to ÜL) (https://www.vr.se/english.html), and from IMI-COMPACT (https://www.imi.europa.eu), the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115363 resources of which are composed of financial contribution from the European Union's Seventh Framework Program (https://ec.europa.eu/research/fp7/index_en.cfm) (FP7/2007-2013) (IMI) and EFPIA companies in kind contribution, the Swedish Cancer Foundation (CAN 2014/259 to ÜL) (https://www.cancerfonden.se), and from the European Regional Development Fund through the Center of Excellence in Molecular Cell Engineering (2014–2020.4.01.15–0013 to ÜL) (https://ec.europa.eu/regional_policy/en/funding/erdf/), and by the Estonian Research Council (IUT20-26 to ÜL) (https://www.etag.ee/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.