In order to improve polymeric gene delivery efficiency, it is desirable to understand how polymer-DNA complexes overcome important intracellular barriers. Polyplexes are typically internalized by an endocytic mechanism such as receptor-mediated endocytosis or macropinocytosis. The polyplexes must be subsequently transported from the cell periphery to the perinuclear region, escape the endocytic vesicle, and gain nuclear entry. Small-molecule inhibitors are available that can specifically interfere with a variety of cellular processes. Our goal was to probe intracellular transport of polyethylenimine (PEI)/DNA polyplexes by disrupting microtubules (MTs) using colchicine, stabilizing MTs using paclitaxel, and destabilizing actin filaments with cytochalasin D, inhibiting dynein using vanadate ions and erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), and inhibiting kinesin with Rose Bengal lactone and adenylylimidodiphosphate (AMP-PNP).

Disruption of microfilaments by cytochalasin D decreased the delivery efficiency of PEI polyplexes 60-80%, though the drug did not significantly inhibit uptake. Depolymerization of microtubules by colchicine decreased transfection efficiency by 75%. Microtubule stabilization with paclitaxel, however, facilitated a 20-fold increase in gene expression. Inhibition of dynein with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by kinesin inhibition by RBL (80%) and AMP-PNP (98%). In all cases, controls confirmed that the observed changes in gene delivery efficiency were not due to changes in polyplex uptake, toxicity or non-specific down-regulation of metabolic activity or gene expression.

In summary, while MT and, surprisingly, microfilament disruption reduced transgene delivery, stabilization of microtubules dramatically improved gene expression. Inhibition of molecular motor proteins dynein and kinesin both reduced gene delivery, suggesting that bi-directional movement of endocytic vesicles is important. By understanding the preferred pathways and cellular mechanisms exploited by polyplexes, we may be able to design more efficient vector materials.