Stable and Efficient Transfection of siRNA for Mutated KRAS Silencing Using Novel Hybrid Nanoparticles A. Lakshmikuttyamma, † Y. Sun, ‡ B. Lu, ‡ A. S. Undieh, § and S. A. Shoyele* ,† † Department of Pharmaceutical Sciences, School of Pharmacy, and ‡ Division of Molecular Radiation Biology, Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, United States § Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, The City College of New York, The City University of New York, 160 Convent Avenue, New York, New York 10031, United States ABSTRACT: siRNA is currently the most widely studied form of RNAi, and it has promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composed of human IgG and poloxamer-188, a stealth polymer, will efficiently deliver mutated KRAS siRNA to A549 cells, leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved, which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA/nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown. KEYWORDS: mutated KRAS, RNAi, hybrid nanoparticles, nuclease, lung cancer, gene knockdown ■ INTRODUCTION RNA interference (RNAi) has been found to be a very effective tool in the knockdown of specific oncogenes in cancer cells. siRNA is currently the most widely studied form of RNAi, and it has a promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. 1 Nevertheless, challenges still occur in the develop- ment of this modality of treatment due to its susceptibility to enzymatic degradation in blood, nonspecific uptake by cells, and the difficulty involved in the transfection of siRNA to cells due to its relatively large size and polarity. 2,3 Clearance by the reticuloendothelial system (RES) is another limiting factor affecting the possible therapeutic application of siRNA. 4-6 To achieve an efficient knockdown by siRNA, different types of delivery systems have been investigated. While viral vectors have been known to transduce cells efficiently, their use in translational medicine has been limited by the possibility of viral toxicity and immunogenic and inflammatory reactions. 7 Nonviral vectors such as lipid-based nanoparticles, polymer- based nanoparticles, mesoporous silica, and protein based nanoparticles are currently being investigated as possible delivery systems for efficient siRNA transfection. 8-11 In order to achieve an efficient delivery of siRNA, the delivery system must have the following properties: protection of siRNA from nuclease degradation during transportation in systemic circulation; minimal RES uptake thereby allowing for long blood circulation time; effective endosomal escape following internalization by host cells; and most important, it must not elicit immunological and inflammatory reaction. 8,11 Lipid nanoparticles, which to date are the most advanced delivery system for siRNA and have shown therapeutic efficacy in clinical trial, 12 have presented with some limitation during recent studies. A recent study revealed that siRNA delivery by lipid-based nanoparticles has been found to be substantially reduced as approximately 70% of the internalized siRNA undergoes exocytosis through egress of the lipid nanoparticles from late endosomes and lysosome. It was then proposed that siRNA delivery efficiency might be improved by designing delivery vehicles that have the ability to escape the recycling pathways. 13 Nanoparticles of poly(D,L-lactide-co-glycolide (PLGA), a biodegradable and biocompatible polymer, are the most widely used polymer-based nanoparticles for siRNA delivery. 14 The major limitation of these nanoparticles is that Received: August 1, 2014 Revised: October 20, 2014 Accepted: October 23, 2014 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A dx.doi.org/10.1021/mp500525p | Mol. Pharmaceutics XXXX, XXX, XXX-XXX