Efficient gene silencing with siRNA formulated in DOP-DETA-based lipid nanoparticles

Development of small interfering RNA (siRNA) therapeutics is a promising approach to address unmet medical needs. However, because RNA is easily degraded by RNases and hardly internalized into the cells, a suitable delivery... [ view full abstract ]

Development of small interfering RNA (siRNA) therapeutics is a promising approach to address unmet medical needs. However, because RNA is easily degraded by RNases and hardly internalized into the cells, a suitable delivery system is required to develop siRNA therapeutics. Development of a siRNA delivery vehicle is the most urgent and crucial thing for clinical success of siRNA-based therapies. In the present study, we designed and synthesized a novel polycationic lipid derivative, a dioleoylphosphate-diethylenetriamine conjugate (DOP-DETA) for the purpose of efficient siRNA delivery. Polycationic liposomes composed of DOP-DETA, cholesterol, and dipalmitoylphosphatidylcholine (DPPC) were prepared and mixed with siRNA to obtain lipid nanoparticles (LNP) loaded with siRNA. To evaluate the delivery potential of the LNP, a reporter gene silencing assay was performed using human fibrosarcoma cell line HT1080 constitutively expressing green fluorescent protein (GFP). The data showed that the LNP loaded with siRNA for GFP induced gene silencing at the concentration of less than 5 nM of siRNA. The gene silencing efficiency and cellular uptake amount of siRNA were dependent on siRNA/lipid molar ratio and increased with increasing concentration of lipids. Confocal microscopic observation demonstrates that siRNA transfected with the LNP diffusely distributed throughout the cytoplasm of the cells. Next, the LNP loaded with siRNA for poly [ADP-ribose] polymerase 1 (PARP1-siRNA) was prepared to evaluate its potential to suppress the expression of the innate protein. The results showed that knockdown of PARP1 protein was markedly observed when murine Lewis lung carcinoma cells had been transfected with 0.5 nM of PARP1-siRNA. The silencing efficiency of the LNP loaded with PARP1-siRNA was approximately 80-fold higher than that obtained with the common transfection reagents. The present findings suggest that DOP-DETA-based LNP might have excellent potential to deliver siRNA to target cells.