(182t) Quantitation of Zwitterion-like Succinylated Polyethylenimine/DNA Polyplex Intracellular Trafficking Kinetics

An important step towards the rational design of nucleic acid delivery agents is an understanding of their endocytic trafficking kinetics. Cargo uptake kinetics, the trafficking pathway, and the trafficking pathway kinetics vary with the cargo’s material properties. For most nucleic acid delivery materials, trafficking kinetics remain uncharacterized and the relationships between trafficking kinetics and transfection efficiency are unclear.

Polyethyleneimine (PEI) has been a benchmark polymer for delivery of nucleic acids for decades, but suffers from poor transfection efficiency (compared to viruses) and serum instability. Our group has synthesized a class of zwitterion-like succinylated PEI derivatives, or zPEIs, which exhibit enhanced serum stability as well as higher transfection efficiencies compared to unmodified PEI. However, to what extent higher transfection efficiency was due to different trafficking pathway and kinetics was unknown.

The present study applies our recently developed technique for quantifying trafficking kinetics, known as DAB cytometry, to compare the intracellular kinetics of PEI/DNA and zPEI/DNA polyplexes. DAB cytometry relies on the known trafficking kinetics of horseradish peroxidase (HRP). HRP catalyzes a crosslinking reaction between 3,3’-diaminobenzidine and hydrogen peroxide that makes the HRP-occupied vesicle easy to separate and analyze for polyplex content. DAB cytometry provides measures of localization of cargo in endocytic trafficking that are more quantitative than those of confocal microscopy and without the nonspecific effects of chemical inhibitors. The goal is to determine the effects of the succinyl groups on zPEI/DNA polyplex trafficking pathway and kinetics.

We find zPEI/DNA polyplexes are internalized more slowly by HeLa cells and remain longer in early endosomes than conventional PEI-based polyplexes. More specifically, by 30 min after transfection, almost half of intracellular PEI/DNA polyplexes were found in the early endosome, while only a third of zPEI/DNA polyplexes were in the early endosome. Moreover, the intracellular fraction of zPEI/DNA polyplexes in the early endosome increased to and plateaued at 65% 2-3 h after transfection, while the early endosomal fraction of PEI/DNA polyplexes decreased from its early spike to 10% by 3 h post-transfection.

Additional DAB cytometry studies are currently underway to investigate potential differences in the rate and extent each type of polyplex is trafficked to the lysosome and the cytosol. Our hypothesis is that PEI/DNA polyplexes traffic more quickly to the lysosome, whereas a greater fraction of zPEI/DNA polyplexes escape the endosome to the cytosol, bypassing the lysosome and accounting for the higher transfection efficiency.