Polymer Nanocarrier System for Endosome Escape and Timed Release of siRNA with Complete Gene Silencing and Cell Death in Cancer Cells Wenyi Gu, †,‡ Zhongfan Jia, † Nghia P. Truong, † Indira Prasadam, ‡ Yin Xiao,* ,‡ and Michael J. Monteiro* ,† † Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia ‡ Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove Campus, Brisbane, Queensland 4059, Australia * S Supporting Information ABSTRACT: An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U- 2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA. T he ability for small interfering RNA (siRNA) to silence specific biological pathways by interfering with messenger RNA (mRNA) holds great promise in cancer and other disease treatments. 1-3 Cationic polymers and liposomes represent the most widely tested nanocarriers for siRNA delivery. 4,5 The greatest challenges for such delivery systems are the ability of the nanocarrier to first escape the endosome and then release siRNA into the cytosol where it can bind onto RNA-induced silencing complex (RISC) for silencing to take place. 6 Escape from the endosome has been recently shown to be extremely inefficient (1-2%) for liposomes, 7 which would presumably be similar for other cationic delivery vehicles. The cationic charge will also limit the release of free siRNA in the cytosol, and accumulation of these cationic nanocarriers will result in unwanted toxicity, especially when administered in multiple doses. Here, we describe a unique polymer that mimics the influenza virus escape mechanism from the endosome and releases the siRNA in a time-dependent manner through a self- catalyzed degradation 8 of the cationic to an anionic side groups made by single-electron transfer living radical polymerization (SET-LRP). 9,10 This degradation process is independent of both the molecular weight of the polymer and pH of the environment, allowing a predictable release time of siRNA regardless of cellular environment. 11 We target the knockdown of U-2OS cell line by inhibiting the polo-like kinase (PLK1) pathway as an in vitro model for osteosarcoma, a bone cancer disease prevalent in young children with very poor survival rates. 12 We further explore the effects of off-target knockdown using an siRNA not specific for PLK1 but specific for the E6 and E7 oncogenes (i.e., S10) expressed in cervical carcinomas and carcinoma-derived cell lines. 13 Polymer nanocarriers usually incorporate pH buffering groups that act as “proton sponges” at low pH values or bind electrostatically to the negatively charged endosome membrane to facilitate endosome escape. 14,15 Incorporation of amine- based buffering groups advantageously allows strong electro- static binding to negatively charged siRNA but conversely through this strong binding inhibits siRNA release. 16-18 Attempts to overcome the release problem has been through the incorporation of side chains molecules that can degrade and release siRNA upon an external or environmental stimulus, including temperature, 19 pH, 20 redox potential, 21 light, 22 electrical pulse, 23 and enzymatic degradation. 24,25 However, the use of remote triggers due to inaccessibility to the tumor and the variability of environmental triggers between cell lines and even within the same cell line has limited their use, requiring a more general and independent approach to siRNA release. Received: August 6, 2013 Revised: August 27, 2013 Published: August 30, 2013 Communication pubs.acs.org/Biomac © 2013 American Chemical Society 3386 dx.doi.org/10.1021/bm401139e | Biomacromolecules 2013, 14, 3386-3389