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non-specificity [1–4]. To overcome these problems, vectors areused for safe delivery of genetic materials in gene therapy.Generally, vectors can be classified into two types. One of themis a viral vector which is commonly used to deliver genetic materialinto the cells. The viral vectors such as retroviruses, lentiviruses,adenoviruses, and adeno-associated viruses are very effective inachieving high transfection efficiency; however, their availabilityfor therapeuticuse inthehumanbody is limitedbecause ofimmuneresponses, safety problems, high cost, and low transgenic size [5–9]. The other type is non-viral vectors which are preferred as saferalternatives to viral vectors for gene therapy. The viral vectors suchas liposome, protein, and cationic polymer have many advantagesincluding stability, safety, low immune response, and cell targetingproperties [5,10]. Thus in recent years, the interest in non-viralvector is increasing, and active research has been reported.Cationic polymers are widely used as carriers for non-viralgenetic materials delivery [11–13]. They can condense withgenetic materials through electrostatic interaction to formpolyplexes and facilitate the cellular uptake by cells [12,13]. Inaddition, the amine group of polyplexes is quick on the uptake ofthe cell absorbing protons, facilitating the escape of the polyplexesfrom endosome or lysosome through a triggered osmotic swellingeffect. [12,14,15].Chitosan is a linear polysaccharide composed of randomlydistributed b-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylate unit) – a structure very similarto that of cellulose. As such chitosan is one of the major cationicpolymers [16,17]. It is obtained by the alkaline deacetylation ofchitin, which is the second most abundant polysaccharides innature after cellulose. Chitosan forms inter- and intra-molecularhydrogen bonding owing to amine and hydroxyl groups; therefore,it has a rigid crystalline structure [18]. Chitosan has a variousbioactivities due to the abundant primary amino groups in thechitosan main chain. For this reasons, the chitosan is extensivelyused to the biomedical fields such as drug and/or gene delivery andthe industrial fields such as water treatment (e.g. harmful algaecontrol), heavy metal flocculants and functional foods [19].Chitosan is soluble in an acid solution but insoluble at naturaland alkaline pH values because of the pKa value of chitosan ofabout 6.5 [20]. The solubility of chitosan is significantly dependenton the degree of deacetylation (DDA). When DDA of chitosan is40%, chitosan is soluble up to a pH of 9. Whereas DDA of chitosanis 80%, it is soluble only up to a pH of 6.5 [18]. Moreover, themolecular weight (MW) of chitosan and the ionic strength of thesolution influence the solubility of chitosan. Reporting on thechemical properties of chitosan including cationic properties,Sanford pointed out that the high charge density at pH < 6.5 formsgels with polyanions, adheres to negatively charged surfaces,chelates certain transitional metals, and is readily susceptible tochemical modification [21]. During the last decade, chitosan hasbeen extensively used as a gene carrier for gene therapy byapplying the chemical properties described above. It has also beenextensively studied as non-viral derived cationic natural polymersfor a number of pharmaceutical and biomedical applications due toits biocompatibility, biodegradability to normal body constituents,non-toxic, hemostatic, bacteriostatic, fungistatic, spermicidal,anticancerogen, anticholesteremic properties easily susceptibleto chemical modification [21–24]. In addition, chitosan is tightlycondensed with negatively charged genetic materials, protectinggenetic materials against nuclease degradation due to cationicproperty as a positive charge [25,26]. Chitosan/DNA polyplexeshave been reported to transfect into various cell types (e.g., humanembryonic kidney cells (HEK293) [13], cervical cancer cells (HeLacell) [13], primary chondrocytes [27], Chinese hamster ovary cells(CHO-K1) [28], fibroblast cells (NIH 3T3) [29], and epithelioma