Visualization of the Process of a Nanocarrier-Mediated Gene Delivery: Stabilization, Endocytosis, Endosomal Escape and Exocytosis of Genes for Intercellular Spreading

Nanoparticles have been widely applied as gene carrier for improving RNA interference (RNAi) efficiency in medical and agricultural fields. However, the mechanism and delivery process of nanoparticle-mediated RNAi is not directly visualized. Here we synthesized a star polymer (SPc) consisted of a hydrophilic shell with positively-charged tertiary amine in the side chain as a gene nanocarrier, which was taken as an example to investigate the mechanism in gene delivery. The SPc could assemble with dsRNA spontaneously through electrostatic force, hydrogen bond and van der Waals force. Interestingly, the SPc could protect dsRNA from degradation by RNase A, insect hemolymph and immune cells, thus remarkably increasing the stability of dsRNA. Meanwhile, the SPc could efficiently promote the cellular uptake, endosomal escape and active exocytosis for systematic spreading of dsRNA. Transcriptome analysis revealed that the SPc could up-regulate some key genes such as Chc, AP2S1 and Arf1 for activating clathrin-mediated endocytosis. Furthermore, the suppression of endocytosis by a specific inhibitor hindered the cellular uptake of SPc-delivered dsRNA in vitro, and the subsequent RNAi effect was also disappeared in vivo. To our knowledge, our study is the first direct visualization of the detailed cellular delivery process and mechanism of nanocarrier-mediated gene delivery. Above mechanism supports the application of nanocarrier-based RNAi in gene therapy and pest management.