1426 Mol. BioSyst., 2012, 8, 1426–1434 This journal is c The Royal Society of Chemistry 2012 Cite this: Mol. BioSyst., 2012, 8, 1426–1434 Synthesis and evaluation of N-(2,3-dihydroxypropyl)-PEIs as efficient vectors for nucleic acidsw Sushil K. Tripathi, a Santosh Yadav, a Kailash C. Gupta* ab and Pradeep Kumar* a Received 29th December 2011, Accepted 17th February 2012 DOI: 10.1039/c2mb05516c Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by 1 H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of B171–190 nm with a zeta potential of B+33–39 mV. Acid–base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying B2.7 folds higher GFP expression than bPEI and B2.3–3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in B51% cells outcompeting bPEI, Superfectt, Fugenet and Lipofectaminet. Sequential delivery of GFP-specific siRNA resulted in B78% suppression of the target gene compared to B49% achieved by Fugenet. All these results demonstrate the potential of these polymers for in vivo gene delivery. Introduction Among the non-viral gene carriers, cationic polymers are fascinating because of their ease of synthesis concurrent with achievable specific physico-chemical and biological properties of these compounds to suit the special requirements as gene delivery agents that include biocompatibility, DNA binding ability and endosomolytic property. 1–3 A number of cationic polymers have been used for this purpose, 4–7 however, PEI has an advantage over the others since it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. 7,8 The positively charged groups available on the polymer are responsible for DNA binding and condensing it into nanoparticles and also facilitating its cellular uptake while the remaining uncharged amine groups are responsible for endosomolytic buffering and help in escaping the particles from the endosome into the cytoplasm. 7,9–11 Therefore, PEI is considered one of the most effective vectors among the synthetic polycations also due to its proton sponge effect. Conversely, high positive charge density appears to be the primary cause of its marked toxicity, 12,13 thereby limiting its use as a gene delivery vector in vivo. Further, the systemically injected cationic polyplexes interact with serum proteins resulting in their rapid clearance from the blood stream. 14 Therefore, it is envisaged to modify these polyplexes to minimize their toxicity as well as interaction with serum proteins prior to their use for systemic gene delivery. One way of diminishing toxicity and the undesirable interactions of cationic polyplexes with serum proteins is to partially reduce their positive charge by ionic or covalent binding to hydrophilic polymers, viz., polyethylene glycol (PEG) 15,16 and polysaccharides. 17–20 Such types of modifications have led to the improved transfection efficiency and reduced cytotoxicity of these polyplexes. 17–20 Here, we have tried to address the concern of toxicity by reacting bPEI (25 kDa) with varying amounts of glycidol (2,3-epoxy-1- propanol) to form 2,3-dihydroxypropyl- grafted-polyethylenimines a CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi - 110007, India. E-mail: pkumar@igib.res.in; Fax: +91 11 27667471; Tel: +91 11 27662491 b CSIR-Indian Institute of Toxicology Research, M.G. Marg, Lucknow - 226001, U.P., India. E-mail: kcgupta@iitr.res.in; Fax: +91 522 2628227; Tel: +91 522 2621856 w Electronic supplementary information (ESI) available. See DOI: 10.1039/c2mb05516c Molecular BioSystems Dynamic Article Links www.rsc.org/molecularbiosystems PAPER Published on 22 February 2012. Downloaded by Rensselaer Polytechnic Institute on 31/10/2014 13:10:24. View Article Online / Journal Homepage / Table of Contents for this issue