Abstract
RNA interference (RNAi) therapeutics are one of the most promising biological interventions in the efficient management of difficult-to-treat diseases. RNAi is mediated by small interfering RNA (siRNA), which induces specific and highly potent gene silencing. However, intracellular delivery of exogenous, chemically synthesized siRNA to the RNAi pathway in the cytosol remains a challenge, and is fully dependent on technologies that can facilitate cytosolic delivery without undesired side effects. One example is a novel delivery system referred to as lipidoid–polymer hybrid nanoparticles (LPNs), which we recently showed mediates highly efficient and safe gene silencing. Here we describe a double emulsion solvent evaporation method for the preparation of siRNA-loaded LPNs and methodologies employed for their physicochemical characterization and biological performance. A solution of siRNA in aqueous buffer is emulsified by sonication with an organic phase containing lipid and polymer into a primary emulsion. Subsequently, the primary emulsion is emulsified with a secondary water phase containing polyvinyl alcohol by sonication, and the organic phase is evaporated, eventually resulting in LPNs. The physicochemical characterization includes determination of (1) hydrodynamic particle size distribution, (2) zeta potential, (3) siRNA encapsulation efficiency, and (4) practical siRNA loading. The transfection experiments are conducted in a cell-based model system using enhanced green fluorescence protein as reporter. The gene silencing effect is also confirmed at the mRNA level by reverse transcription polymerase chain reaction (RT-PCR). The effect of the siRNA-loaded LPNs on cell viability is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
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References
Uprichard SL (2005) The therapeutic potential of RNA interference. FEBS Lett 579(26):5996–6007
Dizaj SM, Jafari S, Khosroushahi AY (2014) A sight on the current nanoparticle-based gene delivery vectors. Nanoscale Res Lett 9(1):252
Thanki K, Zeng X, Justesen S, Tejlmann S, Falkenberg E, Van Driessche E, Morck Nielsen H, Franzyk H, Foged C (2017) Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: a quality by design-based approach. Eur J Pharm Biopharm 120:22–33
Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, Bacallado SA, Nguyen DN, Fuller J, Alvarez R, Borodovsky A, Borland T, Constien R, de Fougerolles A, Dorkin JR, Narayanannair Jayaprakash K, Jayaraman M, John M, Koteliansky V, Manoharan M, Nechev L, Qin J, Racie T, Raitcheva D, Rajeev KG, Sah DW, Soutschek J, Toudjarska I, Vornlocher HP, Zimmermann TS, Langer R, Anderson DG (2008) A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 26(5):561–569
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Acknowledgments
We gratefully acknowledge the support from the Lundbeck Foundation—Denmark (Grant No. R219-2016-908 and R181-2014-3793) and the Novo Nordisk Foundation—Denmark (Grant No. NNF17OC0026526).
Conflicts of interest: The authors declare no conflicts of interest.
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Thanki, K., Zeng, X., Foged, C. (2019). Preparation, Characterization, and In Vitro Evaluation of Lipidoid–Polymer Hybrid Nanoparticles for siRNA Delivery to the Cytosol. In: Ogris, M., Sami, H. (eds) Nanotechnology for Nucleic Acid Delivery. Methods in Molecular Biology, vol 1943. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9092-4_9
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DOI: https://doi.org/10.1007/978-1-4939-9092-4_9
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