title: Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid).
abstract: Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC.
authors.forename: Zi-Xian
authors.initials: ZX
authors.lastname: Liao
authors.forename: Shu-Fen
authors.initials: SF
authors.lastname: Peng
authors.forename: Yi-Cheng
authors.initials: YC
authors.lastname: Ho
authors.forename: Fwu-Long
authors.initials: FL
authors.lastname: Mi
authors.forename: Barnali
authors.initials: B
authors.lastname: Maiti
authors.forename: Hsing-Wen
authors.initials: HW
authors.lastname: Sung
mesh_terms: D000838:Anions; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D048271:Chitosan; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis*; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D005354:Fibrosarcoma / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D053769:Nanocapsules / Q000737:chemistry*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D014162:Transfection / Q000379:methods*
chemical_list: D000838:Anions; D020099:Coated Materials, Biocompatible; D053769:Nanocapsules; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D004247:DNA; D048271:Chitosan