title: Methylated N-(4-N,N-dimethylaminobenzyl) chitosan for novel effective gene carriers.
abstract: The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM(47)-Bz(42)-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM(43)-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM(47)-Bz(42)-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM(47)-Bz(42)-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM(43)-CS/DNA and CS/DNA were formed at a ratio of above 1. TM(47)-Bz(42)-CS showed superior transfection efficiency to TM(43)-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM(47)-Bz(42)-CS and TM(43)-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM(47)-Bz(42)-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.
authors.affiliation: Faculty of Pharmacy, Silpakorn University, Nakhonpathom, Thailand.
authors.forename: Theerasak
authors.initials: T
authors.lastname: Rojanarata
authors.forename: Maleenart
authors.initials: M
authors.lastname: Petchsangsai
authors.forename: Praneet
authors.initials: P
authors.lastname: Opanasopit
authors.forename: Tanasait
authors.initials: T
authors.lastname: Ngawhirunpat
authors.forename: Uracha
authors.initials: U
authors.lastname: Ruktanonchai
authors.forename: Warayuth
authors.initials: W
authors.lastname: Sajomsang
authors.forename: Supawan
authors.initials: S
authors.lastname: Tantayanon
mesh_terms: D001596:Benzylamines / Q000138:chemical synthesis* / Q000378:metabolism / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000378:metabolism / Q000633:toxicity; D003470:Culture Media / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008745:Methylation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D044967:Serum / Q000378:metabolism; D012995:Solubility; D014162:Transfection / Q000379:methods*
chemical_list: D001596:Benzylamines; D003470:Culture Media; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan