Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides

Helmfors, Henrik; Eriksson, Jonas; Langel, Ülo

doi:10.1016/j.ab.2015.05.023

Cited by 21 publications

(14 citation statements)

References 25 publications

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“…The assay was performed as described in Helmfors et al [15], briefly, the PF14:SCO-complexes (2 μM:0.4 μM) were already present in the wells before 7,000 cells were seeded in each well of white clear bottom 96-well microplates. After 24 h the plates were completely emptied of cell medium and frozen at -80˚C.…”

Section: Library Screeningmentioning

confidence: 99%

“…Using the CPP PepFect 14 (PF14) to deliver an SCO gives up to a 100-fold increase in luciferase expression splice-correction compared to untreated cells [14]. This assay was recently modified by our group in order to allow the use of plates with more and smaller wells to increase the throughput [15].…”

Section: Introductionmentioning

confidence: 99%

“…Here we report a high-throughput screening aiming to find new co-treatment of CPP:ON complexes with small molecule drugs giving a modulation of the delivery efficacy and to discover new signaling pathways involved in the transfection of HeLa pLuc705 cells. We obtained a small-molecule drugs library, a mix of allosteric modulators, agonists, antagonists and inverse agonists with diverse targets, consisting of 264 members (S1 Table) and used this library in conjunction with our recently modified splice-correction assay [15] to identify molecules that affect the delivery efficacy of PF14:SCO complexes. The first goal of this study was to identify new cell surface receptors that would be involved in the recognition and uptake of our complexes.…”

Section: Introductionmentioning

confidence: 99%

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Effect of small molecule signaling in PepFect14 transfection

et al. 2020

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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.

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Section: Library Screeningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effect of small molecule signaling in PepFect14 transfection

et al. 2020

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“…Due to the ability to traverse cell membranes, CPPs are widely used in the cellular delivery of proteins (Ana, Wei-Ming & Chin, 2016), plasmid DNA (Kato et al, 2016), oligonucleotides (Helmfors, Eriksson & Langel, 2015), and liposomes (Huang et al, 2013). Meanwhile, due to their aqueous solubility, and tissue penetration and distribution capabilities, CPPs are also increasingly used for delivery of anticancer drugs (Vivès, Schmidt & Pèlegrin, 2008; Fonseca, Pereira & Kelley, 2009).…”

Section: Introductionmentioning

confidence: 99%

Design of new acid-activated cell-penetrating peptides for tumor drug delivery

Yao

Zhang

et al. 2017

PeerJ

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TH(AGYLLGHINLHHLAHL(Aib)HHIL-NH2), a histidine-rich, cell-penetrating peptide with acid-activated pH response, designed and synthesized by our group, can effectively target tumor tissues with an acidic extracellular environment. Since the protonating effect of histidine plays a critical role in the acid-activated, cell-penetrating ability of TH, we designed a series of new histidine substituents by introducing electron donating groups (Ethyl, Isopropyl, Butyl) to the C-2 position of histidine. This resulted in an enhanced pH-response and improved the application of TH in tumor-targeted delivery systems. The substituents were further utilized to form the corresponding TH analogs (Ethyl-TH, Isopropyl-TH and Butyl-TH), making them easier to protonate for positive charge in acidic tumor microenvironments. The pH-dependent cellular uptake efficiencies of new TH analogs were further evaluated using flow cytometry and confocal laser scanning microscopy, demonstrating that ethyl-TH and butyl-TH had an optimal pH-response in an acidic environment. Importantly, the new TH analogs exhibited relatively lower toxicity than TH. In addition, these new TH analogs were linked to the antitumor drug camptothecin (CPT), while butyl-TH modified conjugate presented a remarkably stronger pH-dependent cytotoxicity to cancer cells than TH and the other conjugates. In short, our work opens a new avenue for the development of improved acid-activated, cell-penetrating peptides as efficient anticancer drug delivery vectors.

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“…Usually, protein reporter assays are used to evaluate the transfection efficiency of the siRNA/PEG-PEI complex, and luciferase is one of the reporter proteins that offer a quick method for measuring siRNA-mediated gene silencing in living cells (Auld et al, 2008; Bartlett and Davis, 2006; Helmfors et al, 2015). Luciferase protein activity is correlated to the amount of luciferase protein in the cell following siRNA transfection by measuring luminescence using luminogenic substrates (Choy et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays

Rheiner

Reichel

Rychahou

et al. 2017

International Journal of Pharmaceutics

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Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(L-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P′), PEG-PLL-PAL (3P′), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P′ caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P′< 2PD < 3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay.

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Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides

Cited by 21 publications

References 25 publications

Effect of small molecule signaling in PepFect14 transfection

Effect of small molecule signaling in PepFect14 transfection

Design of new acid-activated cell-penetrating peptides for tumor drug delivery

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays

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