Carbohydrate Polymers 91 (2013) 508–517 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jou rn al hom epa ge: www.elsevier.com/locate/carbpol Effects of molecular weight and pyridinium moiety on water-soluble chitosan derivatives for mediated gene delivery Warayuth Sajomsang a,∗ , Pattarapond Gonil a , Uracha Rungsardthong Ruktanonchai a , Maleenart Petchsangsai a , Praneet Opanasopit b , Satit Puttipipatkhachorn c a National Nanotechnology Center, National Science and Technology Development Agency, Pathumthani, Thailand b Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand c Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand a r t i c l e i n f o Article history: Received 14 May 2012 Received in revised form 16 August 2012 Accepted 16 August 2012 Available online 24 August 2012 Keywords: Chitosan Quaternized chitosan Methylated N-(3-pyridylmethyl) chitosan chloride Molecular weight Pyridinium/trimethyl ammonium ratio Gene delivery a b s t r a c t The aim of this study is to investigate the effects of molecular weight, the pyridinium/trimethyl ammo- nium (Py/Tr) ratio, the nitrogen atoms (N) in the methylated N-(3-pyridylmethyl) chitosan chloride (M3-PyMeChC)/the phosphorus atoms (P) in DNA (N/P) ratio, and the physicochemical properties of nanopolyplexes on transfection efficiency. The water-soluble chitosan derivative, M3-PyMeChC, was used as a non-viral vector to deliver pEGFP-C2 into human hepatoma (Huh7) cell lines. The results revealed that higher molecular weight M3-PyMeChC was able to form complexes completely with DNA at lower N/P ratios than that with lower molecular weights, which led to higher transfection efficiency. Moreover, the M3-PyMeChC with higher Py/Tr ratios showed superior transfection efficiency at lower Py/Tr ratios at all N/P ratios studied. The highest transfection efficiency for the nanopolyplexes occurred for a molec- ular weight of 82 kDa at a N/P ratio of 5. The results indicated that the hydrophobic effect of pyridinium moiety could enhance gene transfection efficiency, which can be attributed to the dissociation of DNA from nanopolyplexes. High Py/Tr ratios in nanopolyplexes tended to decrease cytotoxicity due to delo- calization of positive charge into a pyridine ring while high N/P ratios and molecular weight increased cytotoxicity. Our results showed that the vector was able to spread the positive charge by delocalizing it into a heterocyclic ring, suggesting to a promising approach to mediate higher levels of gene transfection. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Gene therapy is the insertion of genes into a specific cell and tissue to treat or prevent disease. It may be used to replace a faulty gene or to introduce a new gene whose function is to cure a disease. An ideal gene delivery method needs to meet 3 major criteria: (1) it should protect the transgene against degradation by nucleases in intercellular matrices, (2) it should bring the trans- gene across the plasma membrane and into the nucleus of target cells, and (3) it should have no detrimental effects (Gao, Kim, & Liu, 2007). There are two approaches for gene delivery: viral and non-viral. Viral delivery is a conventional approach because viruses have evolved to infect cells with high efficacy. However, clini- cal trials have underscored the safety risks of viral gene delivery ∗ Corresponding author at: National Nanotechnology Center, Nanodelivery Sys- tem Laboratory, National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand. Tel.: +66 2 564 7100; fax: +66 2 564 6981. E-mail address: warayuth@nanotec.or.th (W. Sajomsang). due to the possibility of causing cancer and death (Green, Langer, & Anderson, 2008). For this reasons, much attention has been focused on the non-viral approach due to its potentials to overcome many inherent challenges of viral vectors. Numerous non-viral gene vectors which have several advantages over their viral coun- terparts, including ease of production, low immune response, the ability to transfer large DNA molecules and potential cell target- ing properties, have been developed for gene delivery (Anderson, 1992; Li & Huang, 2000). However, a disadvantage of non-viral gene vectors is their low transfection efficiencies compared to viral vectors. In order to improve the transfection efficiency, numerous cationic polymers such as polyethyleneimine (PEI), poly(l-lysines) (PLL), poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA), and chitosan (Ch) have been studied for in vitro as well as in vivo applications (Kim et al., 2007). Despite PEI having excellent transfection efficiency, it is not an ideal carrier because it is not biodegradable and it can cause considerable cytotoxicity via necrosis and apoptosis (Boussif et al., 1995). Chitosan is one of the candidates for use as a non-viral vector due to its non- toxic, biocompatible, and biodegradable properties in the human body. Moreover, it has been proposed as a safer alternative to 0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbpol.2012.08.053