Research paper Polyethylenimine nanoparticles as an efficient in vitro siRNA delivery system Surendra Nimesh a, * , Ramesh Chandra a,b a Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India b Department of Chemistry, University of Delhi, Delhi, India article info Article history: Received 16 December 2008 Accepted in revised form 1 April 2009 Available online 10 April 2009 Keywords: Gene silencing Nanoparticles Polyethylenimine siRNA GFP abstract Degradation of mRNA by RNA interference is one of the most powerful and specific mechanism for gene silencing. Owing to this property, siRNAs are emerging as promising therapeutic agents for the treatment of inherited and acquired diseases, as well as research tools for the elucidation of gene function in both health and disease. Here we have explored the potential of polyethylenimine (PEI) to deliver siRNA to mammalian cells. Nanoparticles of PEI were prepared by acylating PEI with propionic anhydride followed by cross-linking with polyethylene glycol-bis(phosphate). The nanoparticles size as revealed by DLS stud- ies was found to be 110 nm and AFM investigations showed spherical and compact complexes with an average size of 100 nm. For electro-neutralization of negative charge of siRNA higher amount of nanopar- ticles was required as compared to native PEI. The siRNA delivery efficiency of nanoparticles was assessed by using siRNA against gene coding for green fluorescent protein (GFP). The gene silencing efficiency of PEI nanoparticles was found to be comparable to commercially available transfecting agent Lipofectin but with reduced cytotoxicity. Ó 2009 Elsevier B.V. All rights reserved. 1. Introduction Recently, RNA interference (RNAi) has emerged as a powerful tool for silencing a target gene in gene therapy. RNAi regulates gene expression in mammalian cells through siRNA, which is a double-stranded RNA molecule having 21–23 bp. In this process the degradation of homologous mRNA by double-stranded RNA (dsRNA) is highly sequence specific. The process of RNAi has been found to be very useful for genetic analysis and is rapidly evolving as a potent therapeutic approach for gene silencing [1,2].The RNAi mechanism involves the cleavage of long dsRNA molecules into 21–23 bp nucleotides called small interfering RNAs (siRNAs) by an endogenous RNase III-like enzyme known as Dicer [3]. The siR- NA forms the RNA induced silencing complex (RISC) on complexa- tion with the ribonuclear proteins, which contains the proteins necessary for unwinding the double-stranded siRNA, binding, and cleaving the target messenger RNA [4]. In mammalian cells, the inhibition of mRNA translation takes place by a sequence non-spe- cific interferon response triggered on exposure to dsRNAs with more than 30 bp in length [5,6]. However, the interferon response remains inactive on inserting short siRNAs of 21–23 bp into mam- malian cells which results in mRNA degradation with great se- quence specificity [1]. Synthetic siRNAs of 21–23 nucleotides have been found to suppress the in vitro endogenous and exoge- nous gene expression in mammalian cells [7]. The successful clinical use of siRNA is limited due to problems such as: (1) rapid enzymatic degradation resulting in a short half-life in the blood; (2) poor cellular uptake; and (3) insufficient tissue bio- availability [8–10]. Because of these limitations, native delivery of siRNA to the cells is not effective. The siRNA can be chemically mod- ified to circumvent these problems but these modifications are also associated with certain disadvantages such as decreased mRNA hybridization, higher cytotoxicity and increased unspecific effects [11]. Therefore, a delivery system is required which can protect and efficiently transport siRNA to the cytoplasm of the target cells. The majority of approaches tested so far have utilized viral vec- tors, but recently, the research on non-viral vectors has gained momentum as they offer several advantages, such as easy manip- ulatibility, stability, safety, low cost and high flexibility regarding the size of transgene delivered [12]. Amongst various delivery sys- tems, polycationic polymers have been preferred over other sys- tems due to their ease of preparation, purification and chemical modification along with the long shelf life [13,14]. Polyethyleni- mine (PEI), a polycationic polymer has emerged as one of the most promising candidates for the development of efficient gene deliv- ery vectors [15–18]. The high molecular weight PEI (800 kDa) has been found to exhibit high transfection efficiency as compared to low molecular weight PEIs, but is also associated with high cyto- toxicity [19–21]. Various mechanisms of gene delivery have been suggested for PEI, amongst which ‘‘proton sponge hypothesis” is most accepted [16]. However, this theory has been challenged re- cently [22]. 0939-6411/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2009.04.001 * Corresponding author. Present address: Department of Chemical Engineering, Ecole Polytechnique of Montreal, Quebec, Canada. Tel.: +91 9911160170; fax: +91 1123816312. E-mail address: surendranimesh@gmail.com (S. Nimesh). European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 43–49 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb