Polyplex was formed by mixing 300 μL of purified BERA/GFP-siRNA (200 μg/mL) and 300 μL bPEI10k solution of different concentrations (25, 50, 75 and 100 μg/mL) through pipetting to determine the optimum ratio between RNA and in vivo-jetPEI. Then, the mixture was stored at room temperature for 15 min. BERA/GFP-siRNA-complexed in vitro-jetPEI was made at an N/P ratio of 8. Five weeks post-inoculation, mice were injected intravenously with in vivo-jetPEI (20 μg/mice). After four consecutive treatments every other day, mice were sacrificed 6 h after the last dose.

Recently we have established a novel approach to produce bioengineered noncoding RNA agents (BERAs) in living cells that carry target RNAi molecules (e.g., siRNA and miRNA) and thus act as "prodrugs". Using GFP-siRNA-loaded BERA (BERA/GFP-siRNA) as a model molecule, this study was to define the in vitro and in vivo knockdown efficiency of BERAs delivered by liposome-polyethylenimine nanocomplex (lipopolyplex or LPP). Compared to in vivo-jetPEI(R) (IVJ-PEI) and polyplex formulations, LPP offered greater protection of BERA/GFP-siRNA against degradation by serum RNases. Particle sizes and zeta potentials of LPP nanocomplex remained stable over 28days when stored at 4 degrees C. Furthermore, comparable levels of BERA/GFP-siRNA were delivered by LPP and IVJ-PEI to luciferase/GFP-expressing human SK-Hep1-Luc-GFP or A549-Luc-GFP cells, which were selectively processed into target GFP-siRNA and subsequently knocked down GFP mRNA and protein levels. In addition, LPP-carried BERA/GFP-siRNA was successfully delivered into xenograft tumors and offered more consistent knockdown of tumoral GFP mRNA level in an orthotopic hepatocellular carcinoma (HCC) SK-Hep1-Luc-GFP xenograft mouse model, while IVJ-PEI formulation showed larger variation. These findings demonstrated that lipidation of polyplexes improved serum stability of biologic RNAi molecules, which was efficiently delivered to orthotopic HCC tissues to knock down target gene expression.