Polyplexes traffic through caveolae to the Golgi and endoplasmic reticulum en route to the nucleus.

The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.