Harnessing RNA interference (RNAi) to inhibit hepatitis B virus (HBV) gene expression has promising application to therapy. Here we describe a new hepatotropic nontoxic lipid-based vector system that is used to deliver chemically unmodified small interfering RNA (siRNA) sequences to the liver. Anti HBV formulations were generated by condensation of siRNA (A component) with cationic liposomes (B component) to form AB core particles. These core particles incorporate an aminoxy cholesteryl lipid for convenient surface postcoupling of polyethylene glycol (PEG; C component, stealth/biocompatibility polymer) to give triggered PEGylated siRNA-nanoparticles (also known as siRNA-ABC nanoparticles) with uniform small sizes of 80-100 nm in diameter. The oxime linkage that results from PEG coupling is pH sensitive and was included to facilitate acidic pH-triggered release of nucleic acids from endosomes. Nanoparticle-mediated siRNA delivery results in HBV replication knockdown in cell culture and in murine hydrodynamic injection models in vivo. Furthermore repeated systemic administration of triggered PEGylated siRNA-nanoparticles to HBV transgenic mice results in the suppression of markers of HBV replication by up to 3-fold relative to controls over a 28 day period. This compares favorably to silencing effects seen during lamivudine treatment. Collectively these observations indicate that our PEGylated siRNA-nanoparticles may have valuable applications in RNAi-based HBV therapy.

Recently, we reported for the first time the development of pH-triggered nanoparticles for the functional delivery of small interfering RNA (siRNA) to liver for treatment of hepatitis B virus infections in vivo. Here, we report on systematic formulation and biophysical studies of three different pH-triggered nanoparticle formulations looking for ways to improve on the capabilities of our previous nanoparticle system. We demonstrate how pH-triggered, PEGylated siRNA nanoparticles stable with respect to aggregation in 80% serum can still release siRNA payload at pH 5.5 within 30 min. This capability allows functional delivery to cultured murine hepatocyte cells in vitro, despite a high degree of PEGylation (5 mol %). We also demonstrate that pH-triggered, PEGylated siRNA nanoparticles typically enter cells by clathrin-coated pit endocytosis, but functional delivery requires membrane fusion events (fusogenicity). Biodistribution studies indicate that >70% of our administered nanoparticles are found in liver hepatocytes, post intravenous administration. Pharmacodynamic experiments show siRNA delivery to murine liver effecting maximum knockdown 48 h post administration from a single dose, while control (nontriggered) nanoparticles require 96 h and two doses to demonstrate the same effect. We also describe an anti-hepatitis C virus (HCV) proof-of-concept experiment indicating the possibility of RNAi therapy for HCV infections using pH-triggered, PEGylated siRNA nanoparticles.