TY - JOUR
T1 - Preparation, Characterization, and In Vitro Evaluation of Lipidoid-Polymer Hybrid Nanoparticles for siRNA Delivery to the Cytosol
AU - Thanki, Kaushik
AU - Zeng, Xianghui
AU - Foged, Camilla
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
U2 - 10.1007/978-1-4939-9092-4_9
DO - 10.1007/978-1-4939-9092-4_9
M3 - Journal article
C2 - 30838614
SN - 1064-3745
VL - 1943
SP - 141
EP - 152
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -