Insulin blood level in negative nanoparticles was higher than positive ones. Tight junctions almost disappeared in negative nanoparticles group. Opposite charged insulin:CMCS/CS-NGs had same effect on claudin-4 protein expression. A significant down-regulation of phosphorylated claudin-4 in negative nanoparticles group. Negative nanoparticles triggered stronger paracellular permeability than positive ones.