Preparation, Characterization, and In Vitro Evaluation of Lipidoid-Polymer Hybrid Nanoparticles for siRNA Delivery to the Cytosol.
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.
['Thanki|Kaushik|K|', 'Zeng|Xianghui|X|', 'Foged|Camilla|C|']
D045744:Cell Line, Tumor D002470:Cell Survival D003600:Cytosol D004655:Emulsions D006801:Humans D008055:Lipids D053758:Nanoparticles D010316:Particle Size D011142:Polyvinyl Alcohol D034622:RNA Interference D034741:RNA, Small Interfering D013010:Sonication D014162:Transfection
Small interfering RNA Double emulsion solvent evaporation method Antisense oligonucleotides Lipidoids Lipid–polymer hybrid nanoparticles Poly(D,L-lactic-co-glycolic acid) (PLGA)