Colloids and Surfaces B: Biointerfaces 109 (2013) 197–203 Contents lists available at SciVerse ScienceDirect Colloids and Surfaces B: Biointerfaces jou rn al hom epage: www.elsevier.com/locate/colsurfb Synthesis and characterization of N-ethyl-N’- (3-dimethylaminopropyl)-guanidinyl-polyethylenimine polymers and investigation of their capability to deliver DNA and siRNA in mammalian cells Manohar Mahato, Ashwani K. Sharma ∗ , Pradeep Kumar ∗ Nucleic Acids Research Laboratory, CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi, 110 007, India a r t i c l e i n f o Article history: Received 22 October 2012 Received in revised form 9 January 2013 Accepted 26 March 2013 Available online 9 April 2013 Keywords: Polyethylenimine Transfection Cytotoxicity siRNA Buffering capacity a b s t r a c t Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treatment for various acquired and inherited diseases. Here, we report on the synthesis and characterization of N-ethyl-N’-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine (sGP) polymers and investigation of their capability to carry DNA and siRNA in vitro. Zinc triflate-mediated activation of primary amines of branched polyethylenimine (bPEI) followed by reaction with varying amounts of N-ethyl-N’-(3- dimethylaminopropyl) carbodiimide (EDAC) resulted in the generation of a small series of trisubstituted guanidinyl-modified polyethylenimine polymers. Determination of primary amines on modified poly- mers by TNBS assay revealed 62–84% of the attempted conjugation of EDAC onto bPEI. These modified polymers were shown to condense plasmid DNA and retard its mobility on 0.8% agarose gel. Further, these polymers were evaluated for their capability to carry pDNA into the cells by performing transfection assay on various mammalian cells. All the modified polymer/pDNA complexes exhibited significantly higher levels of gene expression with one of the complexes, sGP3/pDNA complex, displayed ∼1.45 to 3.0 orders of magnitude higher transfection efficiency than that observed in the native bPEI and the commercial transfection reagent, Lipofectamine TM . The efficacy of sGP3 polymer was further assessed by siRNA deliv- ery, which resulted in ∼81% suppression of the target gene. In conclusion, these studies demonstrate the potential of these substituted guanidinyl-modified PEIs as efficient gene delivery vectors. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Non-viral vectors have received increasing interest as safe and efficient carriers of nucleic acids intracellularly [1–4]. These vec- tors employ synthetic chemical materials, such as cationic lipids, polymers, dendrimers, peptides, etc., to deliver the genes of inter- est to the interiors of the target cell [5–9]. These materials interact with negatively charged nucleic acids to form compact nanoparti- cles and provide protection to them from degradation by nucleases. These particles are efficiently taken up by the cells and resulted in the higher level of gene expression [7,10,11]. Among these vec- tors, cationic polymers have shown promising results in nucleic acid delivery. Of these, branched polyethylenimine (bPEI) has been the most efficient and widely used polymer for such applications. The prominent features of bPEI are its intrinsic proton sponge prop- erty and high charge density comprising of 1 ◦ , 2 ◦ and 3 ◦ amines in ∗ Corresponding authors. Tel.: +91 11 27662491; fax: +91 11 27667471. E-mail addresses: ashwani@igib.res.in (A.K. Sharma), pkumar@igib.res.in, pkumar@igib.in (P. Kumar). 1:2:1 ratio, which enable it to condense nucleic acids into small- sized polyplexes that are effectively endocytosed by the cells. The proton sponge property facilitates the release of the polyplexes into the cytosol, which are further transported into the nucleus. High molecular weight bPEI exhibits high transfection efficiency, how- ever, associated with high cytotoxicity due to high charge density (in particular, high density of primary amines) and has a tendency to interact non-specifically with the blood components, which have hampered its clinical applications [12,13]. In order to address these concerns, various modifications have been suggested. Hydrophilic modifications reduce the toxicity by lowering the charge density but also affect the transfection efficiency due to diminished inter- actions with the cell membranes that cause lower uptake and internalization [14]. Alternatively, hydrophobic modifications have also been shown to significantly improve the cell viability and gene delivery efficiency by enhancing pDNA condensation through cooperative binding as well as promoting interactions with the lipophilic cell membranes that facilitate pDNA release for transgene expression [15]. Therefore, a large number of ligands have been conjugated with cationic polymers [16–22]. In such studies, a rela- tively higher amount of modified cationic polymers are required to 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.03.052