International Journal of Pharmaceutics 385 (2010) 194–202 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical Nanotechnology PEI-alginate nanocomposites: Efficient non-viral vectors for nucleic acids Soma Patnaik, Mohammed Arif, Atul Pathak, Naresh Singh, K.C. Gupta ∗,1 Institute of Genomics and Integrative Biology (CSIR), Delhi University Campus, Mall Road, Delhi 110007, India article info Article history: Received 22 April 2009 Received in revised form 20 October 2009 Accepted 20 October 2009 Available online 27 October 2009 Keywords: Branched PEI (25 kDa) Alginic acid GFP Transfection Cytotoxicity siRNA abstract Branched polyethylenimine (PEI, 25 kDa) was ionically interacted with varying amount of alginic acid to block different proportion (2.6–5.7%) of amines in PEI to form a series of nanocomposites, PEI-Al. These nanocomposites, upon interaction with DNA, protected it against DNase I. Among various complexes evaluated, PEI-Al(4.8%)/DNA displayed the highest transfection efficiency in HEK293, COS-1 and HeLa cells that was ∼2–8-folds higher than Superfect TM , Fugene TM , PEI (750 kDa)-Al(6.26%) and PEI alone. The projected nanocomposites were nearly non-toxic to cells in vitro.Furthermore, the concentration of PEI-Al(4.8%) needed to deliver GFP-specific siRNA in COS-1 cells was 20 times lower than PEI (750 kDa)- Al(6.26%). Intracellular trafficking of PEI-Al(4.8%) with or without complexed DNA in HeLa cells shows that both appear in the nucleus after 1 h. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Increasing identification of genetic disorders has raised the pos- sibility of cure by way of gene therapy involving target-specific delivery of therapeutic genes. So far, viral vectors, used to deliver genes, are efficient, but are plagued with safety issues (Servos, 1987; Yang et al., 1994; Miller and Vile, 1995). In contrast, non-viral vectors are preferable to viral vectors due to their reduced toxicity and lower immunogenicity and are easier to design and synthe- size. The classes of polycationic polymers constitute such attractive candidates owing to their ability to condense DNA into nano-size polyplexes (Felgner et al., 1997). Although efficient as gene delivery vectors, cationic polymers are often highly toxic and not permis- sible for use in humans. Systemically injected cationic polyplexes interact with serum proteins resulting in their rapid clearance from the bloodstream (Nishikawa et al., 1998; Dash et al., 1999). There- fore, it is necessary to modify these polyplexes to eliminate their toxicity as well as interaction with serum proteins, before being used for systemic gene delivery. One way to diminish toxicity and avoid the undesirable inter- actions of cationic polyplexes with serum proteins is to reduce positive charge by ionic or covalent binding to hydrophilic poly- ∗ Corresponding author at: Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110007, India; Indian Institute of Toxicology Research, M.G. Road, Lucknow 226 001, India. Tel.: +91 11 27662491; fax: +91 11 27667471. E-mail address: kcgupta@igib.res.in (K.C. Gupta). 1 Indian Institute of Toxicology Research, M.G. Marg, Lucknow 226001, India. mers such as polyethylene glycol (PEG) and polysaccharides (Choi et al., 1998; Baszkin and Norde, 2000). However, due to their functional groups, polysaccharides offer better option than PEG, which does not possess reactive groups required for ligand cou- pling (Erbacher et al., 1999; Passirani et al., 1998). These functional groups present in the polysaccharides can be utilized to graft lig- ands to achieve target-specific delivery (Lemarchand et al., 2004). Alginic acid, a polysaccharide, popularly used in food and phar- maceutical industries (Dumitriu, 1996; Jiang et al., 2007) is used for cell immobilization and encapsulation due to its biocompatibility and gelation with divalent cations (Goosen, 1992). The nanocom- posites of PEI (750 kDa) and alginic acid were explored earlier in our laboratory (Patnaik et al., 2006). The presence of alginic acid in nanocomposites not only decreased the toxicity but also efficiently transfected mammalian cells. Encouraged by these promising results, we thought of replacing PEI (750 kDa) in nanocomposites with PEI (b 25 kDa) as it is considered to be a gold standard (Zwiorek et al., 2004), which may further augment the transfection efficiency of the nanocomposites. Therefore, in continuation with our earlier studies, we prepared a series of PEI (b 25 kDa)-Al nanocompos- ites, evaluated them for transfection efficiency and cytotoxicity in various cell lines and compared with those of commercial transfec- tion reagents. The transfection efficiency evaluated in various cell lines was found to be much superior to PEI (750 kDa)-Al(6.26%), PEI alone, Superfect TM and Fugene TM . Cell cytotoxicity of the pro- jected nanocomposites was almost negligible. We then studied the uptake and intracellular trafficking of labeled PEI (25 kDa)-Al(4.8%) nanocomposite in HeLa cells at different time points. We further showed that PEI (25 kDa)-Al(4.8%) nanocomposite effectively pro- tected complexed DNA for 2 h in vitro. 0378-5173/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2009.10.041