Revisiting the complexation between DNA and polyethylenimine – when and where –S–S– linked PEI is cleaved inside the cell

Abstract

As a non-viral gene vector, long PEI chains are more effective but also more cytotoxic. To solve this problem, people have tried to use disulfide (–S–S–) to link short PEI chains into a long one to generate highly efficient and less cytotoxic gene vectors because –S–S– is degradable inside the cell. In order to investigate when and where –S–S– is cleaved during intracellular trafficking, we designed and synthesized rhodamine B labeled linear PEI chains (M_n ≈ 3 kg mol⁻¹) with one end modified with a mercapto-group so that they can be coupled together via one disulfide bond in the middle and one rhodamine B molecule on each side of the disulfide bond, where fluorescence is self-quenched because two rhodamine B molecules are closely linked together. The cleavage of the –S–S– bond separates the two rhodamine B molecules and enhances their fluorescent intensity. In addition, plasmid DNA was also modified with bodipy, a FRET donor of rhodamine B. Using this specially prepared PEI, we studied the intracellular trafficking of the PEI/DNA polyplexes by using flow cytometry and confocal laser scanning microscopy. Our results reveal that (1) DNA is gradually dissociated from the polyplex before the disulfide bond's cleavage; (2) some of polyplexes escaped from endosomes before reaching lysosomes; and (3) the disulfide bonds are mainly cleaved inside lysosomes at ∼5 h post-transfection.