Abstract
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.
Document Type
Article
Publication Date
8-7-2017
Digital Object Identifier (DOI)
https://doi.org/10.1016/j.ijpharm.2017.06.056
Funding Information
This work was supported by the University of Kentucky Cancer Nanotechnology Training Center (UK CNTC), funded by the NCI/NIH and part of the National Cancer Institute Alliance. This work was also supported by the University of Kentucky Markey Cancer Center pilot grant.
Repository Citation
Rheiner, Steven; Reichel, Derek Alexander; Rychahou, Piotr G.; Izumi, Tadahide; Yang, Hsin-Sheng; and Bae, Younsoo, "Polymer Nanoassemblies with Hydrophobic Pendant Groups in the Core Induce False Positive siRNA Transfection in Luciferase Reporter Assays" (2017). Pharmaceutical Sciences Faculty Publications. 127.
https://uknowledge.uky.edu/ps_facpub/127
Notes/Citation Information
Published in International Journal of Pharmaceutics, v. 528, issues 1-2, p. 536-546.
© 2017 Elsevier B.V. All rights reserved.
This manuscript version is made available under the CC‐BY‐NC‐ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/.
The document available for download is the author's post-peer-review final draft of the article.