Design of a Novel PEGylated Liposomal Vector for Systemic Delivery of siRNA to Solid Tumors.

A small interfering RNA (siRNA) delivery system using dioleylphosphate-diethylenetriamine conjugate (DOP-DETA)-based liposomes (DL) was assessed for systemic delivery of siRNA to tumors. DL carrying siRNA capable of inducing efficient gene silencing with low doses of siRNA were modified with polyethylene glycol (PEG-DL/siRNA) for systemic injection of siRNA. The biodistribution of DL and siRNA in the PEG-DL/siRNA was studied by using radiolabeled DL and fluorescence-labeled siRNA, respectively. DL in the PEG-DL/siRNA showed a high retention in the plasma, accumulation in the tumor, and low accumulation in the liver and spleen after intravenous injection. The in vivo effects of PEGylation were observed only when distearoylphosphatidylethanolamine (DSPE)-PEG but not distearoylglycerol (DSG)-PEG were used. This result suggests that the electrostatic interaction between lipid molecules on the surface of PEG-DL/siRNA was a critical determinant for the in vivo effect of PEGylation. When PEG-DL/siRNA (0.1 mg/kg siRNA) was intravenously injected into tumor-bearing mice, in vivo gene silencing was observed in subcutaneous tumors. These results indicate that PEG-DL/siRNA designed in this study is a promising formulation for systemic use of siRNA.

Key determinants of siRNA delivery mediated by unique pH-responsive lipid-based liposomes.

Lipid-based nanoparticles, a potential nonviral vector due to their good biocompatibility and biodegradability, have been extensively developed for the delivery of small interfering RNA (siRNA). We designed a unique pH-responsive lipid derivative, a dioleylphosphate-diethylenetriamine conjugate (DOP-DETA). DOP-DETA consists of a pH-responsive triamine and unsaturated fatty acids that accelerate membrane fusion. Our results showed that DOP-DETA-based liposomes (DL) efficiently delivered siRNA into the cytoplasm and induced RNA interference even at a low siRNA concentration. The knockdown efficiency of DL depended on the molar ratio of total DL lipids to siRNA. When siRNA was formulated with a sufficient amount of DL, it was efficiently taken up by cells and induced effective gene silencing. Time-lapse imaging showed that siRNA transfected with DL was rapidly internalized into the cells and uniformly dispersed in the cytoplasm within a few minites. The results also showed that DL induced sufficient change in surface charge to allow it to interact with the cell membrane and to allow for rapid endosomal escape. Uptake pathway and time-lapse imaging studies revealed that siRNA was delivered by DL into the cytoplasm, possibly through both macropinocytosis and membrane fusion. The present results emphasize that the modulation of surface charge on nanoparticles is crucial for each siRNA delivery process. Our results also suggest that DL is a potentially useful vector for inducing gene silencing with low-doses of siRNA.