Reducible DNA nanoparticles enhance in vitro gene transfer via an extracellular mechanism

We developed polylysine based DNA nanoparticles (DNA NPs) that contain disulfide linkage in the carrier and demonstrated that this reducible DNA NP enhances in vitro gene transfer via an extracellular mechanism. Polylysine was conjugated through an N-terminal cysteine to a polyethylene glycol chain (PEG) by either a disulfide bond (SS) or a thioether bond (CS), and the resulting PEG-peptide conjugates were used to compact plasmid DNA into reducible SS-DNA NPs or non-reducible CS-DNA NPs with identical physical properties. SS-DNA NPs mediated more than 10-fold higher in vitro gene transfer. Others have suggested that disulfide bonds in synthetic gene carriers undergo cleavage in the reducing environment inside the cell, allowing increased intracellular DNA release. In this study, however, both higher cellular uptake of SS-DNA NPs and inhibition of SS-DNA NP mediated in vitro gene transfer by blocking extracellular free thiols suggested an extracellular mechanism. DePEGylation of SS-DNA NPs by extracellular thiols caused aggregation which might lead to higher cellular uptake and higher transgene expression. A series of SS-DNA NPs prepared with stabilized disulfide bonds survived the extracellular environment without aggregation but lost the superior gene transfer ability, indicating that, in our system, intracellular mechanisms are not involved. These results provided further insight into the mechanisms of in vitro gene transfer enhancement by introducing reducible linkages, contributing to the rational design of more efficient non-viral gene delivery systems.