Chronic or acute hepatitis B and C virus infection often progresses towards fulminant hepatitis, which is associated with high level of morbidity and mortality, which represents a major unmet medical need. Given the known role of Fas in triggering hepatocyte death, we developed a tissue and gene-specific silencing approach using targeted nanoparticles bearing siRNA, which was capable of successfully targeting hepatocytes, inducing gene silencing, and inhibiting pathological changes associated with the hepatotoxic process.
The major challenge in development of siRNA therapeutics is delivery methods to the target cells in vivo. This is caused in part due to poor intracellular uptake and limited blood stability of siRNA. In order to achieve hepatocyte-targeted delivery of Fas siRNA and improved gene transfer by intravenous injection of a liposomes complex, some modifications were made to the original formulation to increase specifically targeting hepatocytes and encapsulation efficiency.
On the basis of previous reported that the galactose is a ligand that binds the asialoglycoprotein receptors (ASGP-R), which is specific for hepatocytes with a high affinity and a rapid internalization rate 
, galactosylated lipid was chemically synthesized through link galactose to the distal end of DSPE-PEG. DSPE-PEG is a PEGlyated lipid, was prepared to keep the stabilization of the particles in a physiological condition and prolong the circulation time in vivo 
. Cationic liposomes tend to aggregate in the presence of serum proteins and are taken up by the reticuloendothelial system due to their positive charge content 
. To increase the serum stability of liposomes, the liposome surface was decorated with 10 mol % Gal-DSPE-PEG lipids by the post-insert method, which assures target ligand localization on the external surface of liposomes. Although some researchers reported ability to increase DSPE-PEG2000 concentration to 20 mol % to preventing aggregation induced by serum proteins 
, in our new liposome formulation, we found when increase DSPE-PEG2000 to 20 mol %, the zeta potential of liposomes become negative charged and reduce transfection efficiency is observed.
Protamine is a highly positive charged peptide that acts as nucleic acid condensation reagent. It reported that condensed siRNAs with protamine can increase delivery efficiency 
. Apart from this, we found when lipid film was hydrated with a solution composed of protamine and condensed siRNA, it can achieves more than 90% entrapment efficacy compare with our previous report 
. Correspondingly, the encapsulated siRNA was also sufficient for silencing Fas gene expression.
To develop efficacy and safety hepatocyte targeting siRNA delivery system, the side effect of immune activation with excessive cytokine release and associated inflammatory syndromes should be avoided. Therefore, we tested the levels of IFN-γ, TNF-α as well as IL-6 to investigate the toxicity of the Gal-LipoNP/siRNA complex. As shown in , none of the Gal-LipoNP/siRNA complexes causes significant inflammatory syndromes and hepatitis in mice.
We employed two different delivery approaches to detect the gene silence efficiency. High-volume intravenous (hydrodynamic) injection of naked siRNA is currently the most common method for inducing RNAi in laboratory animals, despite the global gene-silencing effects and lack of feasibility for human application 
. Although Song et al reported that the use of naked Fas siRNA through hydrodynamic injection can protect mice from fulminant hepatitis 
, this treatment required three times administration of Fas siRNA before ConA-induced hepatitis, which may limit therapeutic attractiveness. Water et al 
investigated the behavior of naked siRNA distribution in vivo and reported that naked siRNA preferentially accumulates in the kidneys and is excreted in the urine. Santel et al 
also reported the biodistribution peculiarity of naked Cy3 labeled siRNAs and discovered after 20 min post-injection, the naked siRNAs are predominantly found in the kidney, with no detectable signals in heart, liver, spleen, lung, and pancreas. Furthermore, they reported that naked siRNAs accumulates in the pole and lumen of the proximal tubules and in the urine 5 min after intravenous injection. These studies revealed that there is no selective uptake of siRNA to any cell type of the tissues investigated in vivo, and this is most likely due to instant renal excretion. Consistent with the current literature, we found that the majority of intravenously injected naked siRNA was concentrated in and cleared by the kidney soon after administration (). If naked Fas siRNA was injected for one time and only less than 20% gene silence efficiency can be observed. In contrast, Gal-liposomes were primarily found in the liver at early time points and accumulated in liver until 48 h later can still see red fluorescence. Thus, our results suggest that Gal-liposomes mediated in vivo delivery may endow siRNA with a time-release property. Because siRNA is protected from endogenous nucleases within the liposomes interior and was targeted to hepatocytes through a specific ligands, it is possible that Gal-LipoNP accumulated in the hepatocytes-populated areas of the liver and slowly released their protected siRNA cargo, resulting in a prolonged gene-silencing effect.
In our study significant therapeutic effects were obtained in ConA induced hepatitis by specific silence Fas gene. Fas-mediated apoptosis play an important role in viral and autoimmune hepatitis, alcoholic liver disease 
. The present study focuses on hepatocyte-targeted silencing of Fas gene. The use of Gal-LipoNP and hepatocyte-targeted delivery of siRNA is not only applicable for fulminant hepatitis but also for many other liver diseases such as transplant rejection, hypercholesteremia, and hepatic genetic diseases. Similar strategy can be used to knock down other critical molecules associated with liver fibrosis, inflammation, as well as liver viral infections, such as hepatitis B virus 
or hepatitis C virus 
In summary, our study demonstrates that siRNA could be specific delivered to the liver using galactosylated liposomes. The Gal-liposomes/siRNA complex can efficiently knock down Fas gene expression in liver; the results further clearly demonstrate that the application of Fas siRNA can prevent fulminant hepatitis. Consequently, the use of galactose ligated liposomes delivering siRNA may represent a promising clinical application of RNAi as a powerful tool for molecular therapy of many liver diseases.