Tumor-homing glycol chitosan/polyethylenimine nanoparticles for the systemic delivery of siRNA in tumor-bearing mice Myung Sook Huh a,d,1 , Seung-Young Lee a,1 , Sangjin Park a , Seulki Lee a , Hyunjin Chung a,b , Sojin Lee a,b , Yongseok Choi b , Yu-Kyoung Oh c , Jae Hyung Park d , Seo Young Jeong d , Kuiwon Choi a , Kwangmeyung Kim a, ⁎, Ick Chan Kwon a, ⁎ a Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, South Korea b School of Life Science and Biotechnology, Korea University, 1 Anam-dong, Seongbuk-gu, Seoul 136-701, South Korea c College of Pharmacy, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, South Korea d Departments of Advanced Polymer and Nanopharmaceutical Sciences, Kyung Hee University, Gyeonggi-do 449-701, South Korea abstract article info Article history: Received 15 November 2009 Accepted 16 February 2010 Available online xxxx Keywords: Glycol chitosan Polyethylenimine siRNA Nanoparticle delivery system Tumor-targeting delivery Cancer treatment Here, we designed a new nano-sized siRNA carrier system composed of biocompatible/biodegradable glycol chitosan polymer (GC) and strongly positively charged polyethylenimine (PEI) polymers. In order to make a stable and tumor-homing nano-sized carrier, each polymer was modified with hydrophobic 5β-cholanic acid, and they were simply mixed to form self-assembled GC–PEI nanoparticles (GC–PEI NPs), due to the strong hydrophobic interactions of 5β-cholanic acids in the polymers. The freshly prepared GC–PEI NPs showed a stable nanoparticle structure (350 nm) and they presented a strongly positive-charged surface (ζ potential = 23.8) that is enough to complex tightly with negatively charged RFP-siRNAs, designed for inhibiting red fluorescent protein (RFP) expression. The siRNA encapsulated nanoparticles (siRNA–GC–PEI NPs) formed more compact and stable nanoparticle structures (250 nm) at 1: 5 weight ratio of siRNA to GC–PEI nanoparticles. In vitro RFP expressing B16F10 tumor cell (RFP/B16F10) culture system, the siRNA– GC–PEI NPs presented a rapid time-dependent cellular uptake profile within 1 h. Moreover, the internalized siRNA–GC–PEI NPs lead to specific mRNA breaks down. Furthermore, our new formulation of siRNA–GC–PEI NPs presented a significant inhibition of RFP gene expression of RFP/B16F10-bearing mice, due to their higher tumor-targeting ability. These results revealed the promising potential of GC–PEI NPs as a stable and effective nano-sized siRNA delivery system for cancer treatment. © 2010 Elsevier B.V. All rights reserved. 1. Introduction RNA interference (RNAi) has been focused on as a powerful and potent therapeutic strategy to downregulate target gene expression in clinical applications [1–4]. This is because the highly efficient and specific gene silencing by RNAi is applicable to all classes of molecular targets as a powerful new therapeutic agent that reduces or eliminates undesirable small molecules and proteins. RNAi is fundamentally induced by 21–25 nucleotide double stranded small interfering RNA (siRNA), which become incorporated with the RNA-induced silencing complex (RISC) and guides endonucleolytic cleavage of the comple- mentary target mRNA [4,5]. The advantages of siRNA as a potential therapeutic agent are due to its naturally occurring conserved phenomenon with high specificity, and there is no limitation for its target gene/mRNA. From the proof-of-concept, various target cells and tissues were encountered mRNA silencing study, including tumor and viral infection. Several clinical studies, such as those focused on treatment for age-related macular degeneration (AMD) [6–8] which is known to cause severe and irreversible vision loss, or for respiratory syncytial virus (RSV) [9,10], a common cause of respiratory tract infection, have been performed and the proven treatments are currently awaiting entry to the market. Direct administration of naked siRNA has proven efficacious through local injection. But in spite of recent successful reports of siRNA therapeutic use, there are still obstacles to be solved. For example, naked siRNA is susceptible to nuclease degradation within physiological fluids and is not able to easily penetrate the cell membrane due to its negative-charge. Thus the key to siRNA application is dependent on the development of effective delivery systems to overcome these issues. For the delivery of siRNA in clinical applications, researchers have studied the siRNA delivery vector systems extensively. Although viral vector systems have shown the potential to be the most efficient systems, synthetic nonviral vectors are being targeted as an alternative because less toxicity is created and they are more easily produced than viral vectors for clinical applications [11,12]. Recently, direct conjugations of siRNA to cholesterol [13,14] or protein [15,16] Journal of Controlled Release xxx (2010) xxx–xxx ⁎ Corresponding authors. Tel.: +82 2 958 5912; fax: +82 2 958 5909. E-mail addresses: kim@kist.re.kr (K. Kim), ikwon@kist.re.kr (I.C. Kwon). 1 These authors contributed equally to this paper. COREL-05391; No of Pages 10 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.02.023 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel ARTICLE IN PRESS Please cite this article as: M.S. Huh, et al., Tumor-homing glycol chitosan/polyethylenimine nanoparticles for the systemic delivery of siRNA in tumor-bearing mice, J. Control. Release (2010), doi:10.1016/j.jconrel.2010.02.023 NANOMEDICINE