Balancing Cationic and Hydrophobic Content of PEGylated siRNA Polyplexes Enhances Endosome Escape, Stability, Blood Circulation Time, and Bioactivity <i>in Vivo</i>

Abstract

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-<i>b</i>-[(2-(dimethylamino)ethyl methacrylate)-<i>co</i>-(butyl methacrylate)], PEG-(DMAEMA-<i>co</i>-BMA), was reversible addition–fragmentation chain transfer (RAFT) synthesized with 0–75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and <i>in vivo</i> bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer “50B”) in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. <i>In vitro</i> delivery of siRNA with polymer 50B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0B) NPs (<i>p</i> < 0.05), due to improved stability and a reduced rate of renal clearance. The 50B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0B (<i>p</i> < 0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery <i>in vivo</i>