Chitosan, Carbon Quantum Dot, and Silica Nanoparticle Mediated dsRNA Delivery for Gene Silencing in Aedes aegypti: A Comparative Analysis Sumistha Das, †,‡ Nitai Debnath, †,‡ Yingjun Cui, † Jason Unrine, § and Subba Reddy Palli* ,† † Department of Entomology, University of Kentucky, Lexington, Kentucky 40546, United States ‡ Amity Institute of Biotechnology, Amity University Haryana, Gurgaon 122413, India § Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, United States * S Supporting Information ABSTRACT: In spite of devastating impact of mosquito borne pathogens on humans, widespread resistance to chemical insecticides and environmental concerns from residual toxicity limit mosquito control strategies. We tested three nanoparticles, chitosan, carbon quantum dot (CQD), and silica complexed with dsRNA, to target two mosquito genes (SNF7 and SRC) for controlling Aedes aegypti larvae. Relative mRNA levels were quantified using qRT-PCR to evaluate knockdown efficiency in nanoparticle-dsRNA treated larvae. The knockdown efficiency of target genes correlated with dsRNA mediated larval mortality. Among the three nanoparticles tested, CQD was the most efficient carrier for dsRNA retention, delivery, and thereby causing gene silencing and mortality in Ae. aegypti. KEYWORDS: RNAi, dsRNA, mosquito, SRC, SNF7, CQD, Chitosan M osquitoes transmit pathogens that cause deadly diseases including malaria, yellow fever, chickungunya, dengue, lymphatic filariasis, encephalitis etc. posing a major threat to human population worldwide. 1,2 The popular strategies of mosquito control include insecticides sprays, repellents, and insecticide-treated nets for adult control, and use of insecticides for larval control. However, these methods are gradually losing their effectiveness due to the development of widespread resistance to chemical insecticides 3,4 by mosquitoes and growing environmental concerns from residual toxicity. Use of bacterial proteins as larvicides are found to be effective but they are not highly used because of unpredictable efficacy under different environmental conditions. 5,6 In this context, RNA interference (RNAi) technology is a promising environmental friendly method to control insects by double-stranded RNA (dsRNA) or small interfering RNA (siRNA) triggered post-transcriptional gene silencing. The ability of dsRNA to silence genes was discovered in the nematode, Caenorhabditis elegans. 7 RNAi technologies are being developed to apply this method in crop improvement, pest control and therapeutics. 8-16 Three general strategies have been elucidated for RNAi in insects: microinjection, soaking, and feeding of dsRNA. 17 dsRNA feeding is perhaps the most convenient and cost-effective RNAi approach for insect control. Delivery of dsRNA by expressing them in plants as well as direct feeding are being developed. 9,18 As the efficiency of RNAi in insects (especially those belonging to Lepidoptera, Hemiptera, and Diptera) is low because of dsRNA lower hydrophilicity and net negative charge, poor delivery and uptake efficiency, and sensitivity to nuclease degradation, there are only a few examples on insect control applications of RNAi. 19 The main challenge in widespread use of this technology is to develop inexpensive and reliable dsRNA production and delivery methods. Moreover, the success of RNAi technology largely depends on the stability of dsRNA or siRNA during and/or after delivery. The half-life of naked siRNA in serum ranges from several minutes to about 1 h. 20,21 Such a short half-life of the nucleic acids will not lead to an adequate RNAi response in an organism unless a high dose of dsRNA or siRNA is applied. Another determinant for successful RNAi in Aedes aegypti is lack of dsRNA transporter genes preventing robust systemic RNAi response. 22 In this context, a carrier system is pertinent for delivering dsRNA to target site. Liposomes are being used as a delivery agents to knockdown target genes (e.g., EphA2, FAK, neuropilin-2, or IL-8) in mouse models of cancer diseases. 23 Another class of nanoparticles (NPs), broadly termed as polymeric NPs are a group of solid, biodegradable, colloidal systems that are widely used as drug or gene carrier. 24 During the past few years, NP mediated RNAi is being developed as an Received: June 14, 2015 Accepted: August 20, 2015 Published: August 20, 2015 Letter www.acsami.org © 2015 American Chemical Society 19530 DOI: 10.1021/acsami.5b05232 ACS Appl. Mater. Interfaces 2015, 7, 19530-19535