155. Lipid-Peptide Receptor-Targeted siRNA Nanoparticles for Systemic Delivery To Tumours
Authors: Aristides D. Tagalakis, Vignesh Jayarajan, Ruhina Maeshima, Farhatullah Syed, Stephen L. Hart
Journal: Molecular Therapy (2015)

Studies into the systemic delivery of siRNA to tumours with cationic nanoparticles often report problems of rapid clearance from the circulation, particularly the liver. Our aim was to optimize the surface properties of nanoparticles to retain maximal transfection efficiency while achieving tumour specific siRNA transfection. Here we prepared cationic and anionic receptor-targeted nanocomplexes (RTNs) and compared them for their biophysical properties, biodistribution properties and efficacy in MYCN silencing in vitro and in vivo. RTN formulations comprised a mixture of receptor-targeted peptides and lipids. The peptide contains an RGD integrin-targeting motif, linked to a sixteen-lysine DNA-binding sequence via an RVRR motif, cleavable by endosomal enzymes that promote vector disassembly. In addition, cationic RTNs contained a mixture of lipids including cationic DOTMA, the fusogenic lipid DOPE and stealth lipid DPPE-PEG2000. Anionic formulations were prepared by first preparing a cationic, non-PEGylated RTN core, then adding a final layer of the anionic lipid DOPG along with DOPE and DPPE-PEG2000 to make an LPRL siRNA nanocomplex. Cationic and anionic RTNs were both shown to be spherical with a diameter of 90-120 nm. The anionic PEGylated nanoparticles were the most stable in serum, whereas both anionic and cationic PEGylated RTNs were similarly stable in a saline buffer. MYCN silencing by anionic and cationic PEGylated RTN siRNA transfections in three different neuroblastoma cell lines achieved silencing of 50-90% with no significant differences between the two formulations. MYCN silencing led to reduced cell proliferation (Ki67 staining) and increased apoptosis by more than 7-fold compared to control cells (up to 90% apoptosis). In an in vivo xenograft model of neuroblastoma, mice were injected intravenously with RTNs containing FAM-fluorescently-labelled siRNA. In vivo fluorescence imaging and histological analysis of tissue sections at 4 h and 24 h post injection showed that both the anionic and cationic PEGylated RTNs accumulated in the tumour with trace amounts only detected in the liver and the lung. Intravenous injection of a single dose of RTN with MYCN siRNA (0.64 mg/kg) showed significant levels of silencing for the cationic (30% mean) and anionic formulation (40% mean, with maximum of 63% silencing) compared to control groups. Multiple injections of anionic and cationic PEGylated RTNs showed significant tumour retardation compared to control mice. RTNs overcome a major limitation in the development of siRNA cancer therapies in that, due to their favourable biophysical and surface properties, they avoid significant clearance by the liver allowing accumulation in the tumour by the enhanced permeation and retention effect while their cell targeting and fusogenic properties enable efficient transfection of the target tumour cells, in vitro and in vivo. Anionic RTNs were as efficient and specific as their cationic counterparts, however, anionic nanocomplexes offer the potential advantage of reduced systemic and cellular toxicity. These novel, targeted, RTNs have suitable properties for the development of tumour-specific siRNA therapeutics.