A comprehensive comparative analysis of transfection reagents for siRNA delivery

doi:10.5246/jcps.2024.01.003

Abstract

Abstract:

siRNAs have emerged as essential tools in biological research and hold promise as next-generation therapeutics for gene therapy. However, achieving successful and safe siRNA transfection remains a challenge, especially for cells that are notoriously difficult to transfect. The efficiency of siRNA transfection is influenced by factors such as uptake, cellular availability, and cytotoxicity. Generally, higher transfection efficiency is associated with increased uptake, improved cellular availability, and reduced cytotoxicity. This study aimed to compare the performance of different transfection reagents in delivering siRNAs, focusing on their uptake, knockdown efficacy, and toxicity. All reagents measured in here were good uptaken in HepG2 and HEK293T cells, but low uptake in RAW264.7 cells. Among them, the newly developed CALNP RNAi transfection reagent exhibited distinct uptake profiles when compared to other transfection reagents across various tested cell lines. CALNP RNAi consistently demonstrated superior transfection efficiency and minimal cell toxicity in all the tested cell lines. Furthermore, CALNP RNAi held promise for enabling RNA interference in "hard-to-transfect" cells. This research contributed to our understanding of effective siRNA delivery methods and highlighted the potential of CALNP RNAi transfection reagent as a valuable tool for gene silencing, especially in challenging cellular contexts.

Key words: Transfection reagent, siRNA, RNA interfering, "Hard-to-transfect" cells

Supporting:

Shuwen Tong, Xianrong Qi. A comprehensive comparative analysis of transfection reagents for siRNA delivery[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(1): 26-34.

Figures/Tables 11

References 22

[1]	Rajeev, A.; Siby, A.; Koottungal, M.J.; George, J.; John, F. Knocking down barriers: advances in siRNA delivery. Chem. Select. 2021, 6, 13350–13362.
[2]	Petrilli, R.; Eloy, J.O.; de Souza, M.C.; Abriata Barcellos, J.P.; Marchetti, J.M.; Yung, B.; Lee, R.J. Lipid nanoparticles as non-viral vectors for siRNA delivery. Nanobiomater. Drug Deliv. 2016, 75–109.
[3]	Setten, R.L.; Rossi, J.J.; Han, S.P. The Current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 2019, 18, 421–446.
[4]	Akinc, A.; Maier, M.A.; Manoharan, M.; Fitzgerald, K.; Jayaraman, M.; Barros, S.; Ansell, S.; Du, X.Y.; Hope, M.J.; Madden, T.D.; Mui, B.L.; Semple, S.C.; Tam, Y.K.; Ciufolini, M.; Witzigmann, D.; Kulkarni, J.A.; van der Meel, R.; Cullis, P.R. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 2019, 14, 1084–1087.
[5]	Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997, 23, 3–25.
[6]	Dowdy, S.F. Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 2017, 35, 222–229.
[7]	Rahimi, P.; Mobarakeh, V.I.; Kamalzare, S.; SajadianFard, F.; Vahabpour, R.; Zabihollahi, R. Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents. Bratisl. Lek. Listy. 2018, 119, 701–705.
[8]	Yamano, S.; Dai, J.S.; Moursi, A.M. Comparison of transfection efficiency of nonviral gene transfer reagents. Mol. Biotechnol. 2010, 46, 287–300.
[9]	Nolte, A.; Raabe, C.; Walker, T.; Simon, P.; Ziemer, G.; Wendel, H.P. Optimized basic conditions are essential for successful siRNA transfection into primary endothelial cells. Oligonucleotides. 2009, 19, 141–150.
[10]	Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stöter, M.; Epstein-Barash, H.; Zhang, L.G.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638–646.
[11]	Wittrup, A.; Ai, A.; Liu, X.; Hamar, P.; Trifonova, R.; Charisse, K.; Manoharan, M.; Kirchhausen, T.; Lieberman, J. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 2015, 33, 870–876.
[12]	Yoo, J.W.; Hong, S.W.; Kim, S.; Lee, D.K. Inflammatory cytokine induction by siRNAs is cell type- and transfection reagent-specific. Biochem. Biophys. Res. Commun. 2006, 347, 1053–1058.
[13]	Kleefeldt, J.M.; Pozarska, A.; Nardiello, C.; Pfeffer, T.; Vadász, I.; Herold, S.; Seeger, W.; Morty, R.E. Commercially available transfection reagents and negative control siRNA are not inert. Anal. Biochem. 2020, 606, 113828.
[14]	Danielli, M.; Marinelli, R.A. Lipid-based transfection reagents can interfere with cholesterol biosynthesis. Anal. Biochem. 2016, 495, 1–2.
[15]	Figueroa, E.; Bugga, P.; Asthana, V.; Chen, A.L.; Stephen Yan, J.; Evans, E.R.; Drezek, R.A. A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines. J. Nanobiotechnol. 2017, 15, 1–15.
[16]	Wang, S.; Tian, D. High transfection efficiency and cell viability of immune cells with nanomaterials-based transfection reagent. BioTechniques. 2022, 72, 219–224.
[17]	Jensen, K.; Anderson, J.A.; Glass, E.J. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation. Vet. Immunol. Immunopathol. 2014, 158, 224–232.
[18]	Moore, J.C.; Atze, K.; Yeung, P.L.; Toro-Ramos, A.J.; Camarillo, C.; Thompson, K.; Ricupero, C.L.; Brenneman, M.A.; Cohen, R.I.; Hart, R.P. Efficient, high-throughput transfection of human embryonic stem cells. Stem Cell Res. Ther. 2010, 1, 1–11.
[19]	Ma, Y.H.; Lin, H.F.; Qiu, C.H. High-efficiency transfection and siRNA-mediated gene knockdown in human pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 2012, 21, 5C.2.1–5C.2.9.
[20]	Keller, A.A.; Maeß, M.B.; Schnoor, M.; Scheiding, B.; Lorkowski, S. Transfecting macrophages. Macrophages. 2018, 187–195.
[21]	Herb, M.; Farid, A.; Gluschko, A.; Krönke, M.; Schramm, M. Highly efficient transfection of primary macrophages with in vitro transcribed mRNA. J. Vis. Exp. 2019, e60143.
[22]	Zhang, X.; Edwards, J.P.; Mosser, D.M. The expression of exogenous genes in macrophages: obstacles and opportunities. Macrophages Dendritic Cells. 2009, 123–143.