Polymeric nanoparticles (NPs) have attracted increasing interest as nonviral vector for gene delivery. Among polyion complexes, chitosan(CS)/DNA complex NPs have been considered as a candidate for gene delivery. It was demonstrated that after modification of internal structure of CS/DNA complexes with γ-PGA, cellular uptake and transfection effiency of CS/DNA/γ-PGA NPs were significantly higher. Moreover, DNA/Polyethylenimine complex coated with γ-PGA is effective as a gene delivery system with high transfection efficiency and low toxicity. However, the role of γ-PGA has not been elucidated clearly. In this study, an approach through coating CS/DNA complexes by a negatively charged γ-PGA was reported and the mechanism of enhanced cellular uptake and transfection efficiency of these complexes in the presence of γ-PGA was clarified. Results of particle size, zeta potential and molecular dynamics (MD) simulation indicated that γ-PGA covered the surface of CS/DNA NPs. Furthermore, observation from confocal microscope revealed that free form of γ-PGA and CS/DNA NPs coated by anionic γ-PGA could enter into HT1080 cells. In addition, cellular uptake of CS/DNA complexes was enhanced significantly due to coating of γ-PGA. Taken altogether, it indicated that γ-PGA helped increase significantly their cellular uptake via different uptake mechanism from CS/DNA complexes with a cationic charge. γ-PGA also reduced CS binding to DNA so that DNA could release easily after transfected into the cells, hence transfection efficiencies were enhanced. However, when the quantity of γ-PGA was increased, more and more free amount of negative charge covered the surface of NPs, hence repulsive force between γ-PGA and lipid bilayers became stronger. Therefore, it was difficult for NPs to reach to the cell membrane and resulted in low transfection efficiency. We futher demonstrated that caveolae-mediated routes were main pathways to taken up CS/DNA NPs coated by γ-PGA into the cells. Besides, GGT, a cell membrane bound enzyme, was also concerned with cellular uptake mechanism of ternary complexes via specific interaction with γ-PGA. Finally, crucial role of terminal amine group of γ-PGA on cellular uptake of NPs has been proved. In other words, it might have specific interaction with the cell membrane and helped NPs enter into HT1080 cells easily. Our results resolved the mechanism of enhanced cellular uptake and transfection efficiency of CS/DNA complexes coated by γ-PGA.

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