(568n) Molecular Behaviors of Nucleic Acid/Polymer Nanoparticles Under Intracellular Conditions: In Situ Atomic Force Microscopy Study

Understanding intracellular trafficking of nucleic acid/polymer complexes (polyplexes) is crucial in designing novel nonviral gene delivery vehicles. However, elucidating dynamic nucleic acid/polymer interactions under differentially and sequentially varying intracellular conditions has been a major technical challenge. In this study, using in situ atomic force microscopy (AFM), plasmid DNA and siRNA complexed with various unmodified or acid-degradable polyethylenimine (PEI, branched or linear) were observed in a flow cell that simulates mildly acidic endosomal and reducible cytosolic conditions (i.e., pH 5.0 and heparan sulfate-containing buffers, respectively). Results showed that both DNA and siRNA were more efficiently dissociated from acid-degradable linear PEI than acid-degradable branched one at pH 5.0, while no evidence of nucleic acid disassembly from unmodified PEI (both linear and branched forms) was obtained under the same condition. Unexpectedly, a cytosolic proteoglycan, heparan sulfate, was simply adsorbed onto the cationic polymers rather than disassembling the polyplexes, regardless of polymer types. Finally, the AFM results along with in vitro DNA transfection and RNA interference indicated that rapid dissociation of siRNA significantly enhances gene silencing while DNA needs to be complexed with polymer for efficient transfection. This study proposes new insightful formation for designing efficient nonviral gene carriers by better understanding complexation and disassembly dynamics of nucleic acid and polymer during intracellular pathways.