Quantum-dot-assisted fluorescence resonance energy transfer approach for intracellular trafficking of chitosan/DNA complex.
Fluorescence resonance energy transfer (FRET) was employed to monitor the molecular dissociation of a chitosan/DNA complex with different molecular weights of chitosan. Chitosan with different molecular weights was complexed with plasmid DNA and the complex formation was monitored using dynamic light scattering and a gel retardation assay. As the chitosan molecular weight increased, a more condensed complex was prepared at various ratios of chitosan to DNA. Plasmid DNA and chitosan were separately labeled with quantum dots and Texas red, respectively, and the dissociation of the complex was subsequently monitored using confocal microscopy and fluorescence spectroscopy. As the chitosan molecular weight in the chitosan/DNA complex increased, the Texas red-labeled chitosan gradually lost FRET-induced fluorescence light when HEK293 cells incubated with chitosan/DNA complex were examined with confocal microscopy. This suggests that the dissociation of the chitosan/DNA complex was more significant in the high molecular weight chitosan/DNA complex. Fluorescence spectroscopy also monitored the molecular dissociation of the chitosan/DNA complex at pH 7.4 and pH 5.0 and confirmed that the dissociation occurred in acidic environments. This finding suggests that the high molecular weight chitosan/DNA complex could easily be dissociated in lysosomes compared to a low molecular weight complex. Furthermore, the high molecular weight chitosan/DNA complex showed superior transfection efficiency in relation to the low molecular weight complex. Therefore, it could be concluded that the dissociation of the chitosan/DNA complex is a critical event in obtaining the high transfection efficiency of the gene carrier/DNA complex.
Lee
Jung Im
JI
Department of Biomaterials Engineering, School of Bioscience and Bioengineering, Kangwon National University, Chuncheon, Republic of Korea.
Ha
Kwon-Soo
KS
Yoo
Hyuk Sang
HS
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D048271:Chitosan / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D041681:NIH 3T3 Cells; D010316:Particle Size; D045663:Quantum Dots; D014162:Transfection
D004247:DNA; D048271:Chitosan