In vivo vascular and tumor cell gene silencing with chitosan nanoparticles in ovarian carcinoma

5613 Objective: To develop a novel chitosan nanoparticle based delivery method for in vivo gene silencing in tumor cells as well as the angiogenic vasculature.
 Methods: SiRNA-chitosan (siRNA-CH) nanoparticles were prepared based on ionic gelation of anionic TPP and siRNA. The loading efficiency, size and zeta potential of the siRNA-CH nanoparticles was measured. The delivery of i.v. siRNA-CH into orthotopic ovarian tumors was examined using Alexa-555 conjugated siRNA. Proof-of-concept studies with murine or human siRNA targeted EZH2 were performed in orthotopic mouse models of ovarian carcinoma (HeyA8 and SKOV3ip1) and gene silencing, therapeutic efficacy and biological effects on angiogenesis (MVD) and apoptosis (TUNEL) were examined.
 Results: The siRNA-CH nanoparticles are approximately 100 nm in size and have a slight positive charge. We optimized the incorporation efficiency to achieve >70% siRNA incorporation into chitosan. A single i.v. injection resulted in deep penetration of the siRNA into tumor parenchyma, with >80% of fields with siRNA in tumor cells. SiRNA delivery was clearly noted in most of the tumor endothelial cells. SiRNA uptake was also observed in the heart, liver, kidney and lung tissues. Macrophage staining using f4/80 showed that macrophages have less amount of siRNA compared to tumor cells suggested that siRNA was delivered directly into the tumor cells. For proof-of-concept studies, we selected, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), which we have previously demonstrated as being overexpressed in both tumor and tumor-associated endothelial cells. EZH2 siRNA-CH was effective in decreasing EZH2 levels in the tumor (human sequence) and endothelial cells (mouse sequence) for up to 6 days. EZH2 silencing of this gene using either mouse or human specific siRNA significantly reduced tumor growth in both models (24 to 70% reduction, p values