Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation.
Chitosans of defined molecular weight (Mw 10-213 kDa) and degree of deacetylation (DD 46-88%) were synthesized, complexed with pEGFP-C2 plasmid into nanoparticles (NP) and evaluated for cellular uptake and transfection efficiency in the A549 cell model. DNA condensation of >90% was achieved at the N/P ratio of 6, independent of the chitosan Mw and DD. However, chitosan vectors of lower Mw or DD were less efficient at retaining the DNA upon dilution, and consequentially, less capable of protecting the condensed DNA from degradation by DNase and serum components. A549 cellular uptake of the NP was also significantly reduced by decreasing the Mw or DD of the chitosan vector. These factors contributed to the low transfection efficiencies for chitosan vectors of low Mw or DD. There was good correlation between transfection efficiency, cellular uptake and zeta potential of the NP, suggesting that cellular uptake mediated by electrostatic interactions with the cell membrane preceded efficient transfection. NP produced with chitosan of Mw 213 kDa and DD of 88% showed the highest zeta potential (+23 mV), cellular uptake (4.1 microg/mg protein) and transfection efficiency (12.1%), while chitosan vector with Mw of 213 kDa and DD of 46% showed the lowest cellular uptake (0.4 microg/mg protein) and transfection efficiency (0.05%). Confocal microscopy images suggested that the chitosan-complexed DNA successfully escaped from the endo-lysosomal compartment for nuclear translocation and expression. Intracellular DNA disassembly appeared to occur at different locations depending on the retentive capacity of the chitosan vector.
Huang
Min
M
Department of Pharmacy, National University of Singapore, Singapore.
Fong
Chee-Wai
CW
Khor
Eugene
E
Lim
Lee-Yong
LY
D000107:Acetylation; D048271:Chitosan / Q000008:administration & dosage*; D004247:DNA / Q000008:administration & dosage*; D006801:Humans; D008970:Molecular Weight; D049329:Nanostructures; D014162:Transfection / Q000379:methods*
D004247:DNA; D048271:Chitosan