Enhancement of efficiencies of the cellular uptake and gene silencing of chitosan/siRNA complexes via the inclusion of a negatively charged poly(γ-glutamic acid).
Although advantageous for siRNA packing and protection, chitosan (CS)-based complexes may lead to difficulties in siRNA release once they arrive at the site of action, due to their electrostatic interactions. To assist the intracellular release of siRNA and thus enhance its effectiveness in gene silencing, we incorporated a negatively charged poly(γ-glutamic acid) (γ-PGA) into CS/siRNA complexes. The inclusion of γ-PGA did not alter the complex-formation ability between CS and siRNA; additionally, their cellular uptake was significantly enhanced. The results obtained in our molecular dynamic simulations indicate that the binding between CS and siRNA remained stable in the cytosol environment. In contrast, the compact structure of the ternary CS/siRNA/γ-PGA complexes was unpacked; such a structural unpackage may facilitate the intracellular release of siRNA. In the gene silencing study, we found that the inclusion of γ-PGA into complexes could significantly expedite the onset of gene knockdown, enhance their inhibition efficiency and prolong the duration of gene silencing. These findings may be attributed to the fact that there were significantly more CS/siRNA/γ-PGA complexes internalized into the cells in company with their more rapid intracellular unpackage and release of siRNA when compared with their binary counterparts in the absence of γ-PGA. The aforementioned results suggest that CS/siRNA/γ-PGA complexes can be an efficient vector for siRNA transfection.
Liao
Zi-Xian
ZX
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.
Ho
Yi-Cheng
YC
Chen
Hsin-Lung
HL
Peng
Shu-Fen
SF
Hsiao
Chun-Wen
CW
Sung
Hsing-Wen
HW
D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D018613:Microscopy, Confocal; D056004:Molecular Dynamics Simulation; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism*
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; C511775:poly(gamma-glutamic acid); D049452:Green Fluorescent Proteins; D011099:Polyglutamic Acid; D048271:Chitosan; D008156:Luciferases