Development of a novel Polymer-based delivery system for siRNAs to hepatocellular carcinoma cells

For Hepatocellular carcinoma (HCC) there are no effective treatments thus, the individuation of novel therapeutic approaches and molecular targets are urgent. Small interference RNA (siRNAs) are small double stranded RNAs able to induce gene silencing thus, representing an attractive therapeutic strategy. The main impediment in using siRNAs is the fast degradation rate in the biological environment. To protect siRNAs from degradation, we developed a novel delivery system based on the polymer α,β-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) derivatized with diethylene triamine (DETA) linked via a polyethylene glycol (PEG) to galactose (GAL) molecules (PDPG). The GAL residue has been introduced to target to the asialoglycoprotein receptor (ASGPR), overexpressed in HCC cells. Using a fluorescently labelled PDPG (PDPG-BodipyTRX) carrying a model siRNA, we demonstrated that the PDPG polymer can specifically deliver the siRNA to HuH7 cells, a cellular model of HCC. Notably, the elimination of the Gal residue from the PDPG (PDP) or the chemical block of ASGPR, resulted in an evident reduction of PDPG targeting effectiveness. These data have been confirmed in HuH7 constitutively expressing the enhanced green fluorescence protein (EGFP) using a functional siRNA against EGFP. Finally, PDPG delivery specificity and effectiveness was proved also in vivo in a mouse dorsal skinfold window chamber assay. Together, our data support the remarkable targeting ability of the developed PDPG. When PDPG was loaded by siRNAs targeting HCC proliferative factors (eEF1A1, eEF1A2 and E2F1), we observed a clear decrease of HuH7 cell vitality and cell number. These phenomena were paralleled by the reduction of targeted mRNAs and relative proteins levels. Interestingly, siRNAs-PDP, lacking the Gal residue was poorly effective in reducing HuH7 vitality and number. Finally, in vivo experiments performed in a subcutaneous xenograft mouse model of HCC, revealed the ability of siRNAs-PDGP to reduce HCC tumor growth compared to controls. Notably, PDPG did not show significant polymer-related toxic effects as evaluated by monitoring animal weight and the levels of markers of organs function. In conclusion, our study demonstrates the priceless potentials of PDPG as a novel delivery system for siRNA paving the way for innovative anti-HCC treatments.