Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene delivery vector.
Chitosan (CS)-based complexes have been considered as a vector for DNA delivery; nonetheless, their transfection efficiency is relatively low. An approach by incorporating poly(γ-glutamic acid) (γ-PGA) in CS/DNA complexes was developed in our previous study to enhance their gene expression level; however, the detailed mechanisms remain to be understood. The study was designed to investigate the mechanisms in cellular uptake and intracellular trafficking of CS/DNA/γ-PGA complexes. The results of our molecular dynamic simulations suggest that after forming complexes with CS, γ-PGA displays a free γ-glutamic acid in its N-terminal end and thus may be recognized by γ-glutamyl transpeptidase in the cell membrane, resulting in a significant increase in their cellular uptake. In the endocytosis inhibition study, we found that the internalization of CS/DNA complexes took place via macropinocytosis and caveolae-mediated pathway; by incorporating γ-PGA in complexes, both uptake pathways were further enhanced but the caveolae-mediated pathway played a major role. TEM was used to gain directly understanding of the internalization mechanism of test complexes and confirmed our findings obtained in the inhibition experiments. After internalization, a less percentage of co-localization of CS/DNA/γ-PGA complexes with lysosomes was observed when compared with their CS/DNA counterparts. A greater cellular uptake together with a less entry into lysosomes might thus explain the promotion of transfection efficiency of CS/DNA/γ-PGA complexes. Knowledge of these mechanisms involving CS-based complexes containing γ-PGA is critical for the development of an efficient vector for DNA transfection.
Peng
Shu-Fen
SF
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
Tseng
Michael T
MT
Ho
Yi-Cheng
YC
Wei
Ming-Cheng
MC
Liao
Zi-Xian
ZX
Sung
Hsing-Wen
HW
D045744:Cell Line, Tumor; D002470:Cell Survival; D048271:Chitosan / Q000378:metabolism*; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D018014:Gene Transfer Techniques; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D056004:Molecular Dynamics Simulation; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D021381:Protein Transport; D014162:Transfection
C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D004247:DNA; D048271:Chitosan; D008156:Luciferases