The effect of crosslinking agents on the transfection efficiency, cellular and intracellular processing of DNA/polymer nanocomplexes

Cellular and intracellular processing of DNA/polymer nanocomplexes was optimized by tailoring the composition of crosslinking agents for improving in vitro and in vivo transfection efficiency. Nanocomplexes composed of trimethyl chitosan-arginine conjugate (TMC-Arg), plasmid DNA (pDNA), and different proportions of sodium tripolyphosphate (TPP) and poly(γ-glutamic acid) (γ-PGA) were prepared. All TMC-Arg nanocomplexes (TANC) possessed similar particle sizes and preferable protection of pDNA against degradation. The Zeta potentials of TANC decreased with increasing amount of TPP, which were positively correlated to their cellular uptake levels. The composition of crosslinking agents affected their internalization mechanisms, wherein the addition of γ-PGA changed from clathrin-mediated endocytosis to caveolae-mediated one. The increment of TPP amount in TANC was responsible for their reduced binding affinity to pDNA and rapid pDNA release, which was related to their subcellular distribution and in vitro and in vivo transfection patterns. More compact TANC were associated with a delayed protein expression while easily dissociated ones gave a faster onset of action and higher short term gene transfer. However, TANC that dissociated too readily had the inability of gene transfection owing to pDNA degradation in the endolysosomes. Therefore, tailoring the composition of crosslinking agents in nanocomplexes may provide a feasible tool for improving transfection efficiency.