Biochemical investigation of active intracellular transport of polymeric gene-delivery vectors.
To design safe, efficient synthetic gene therapy vectors, it is desirable to understand the intracellular mechanisms that facilitate their delivery from the cell surface to the nucleus. Elements of the cytoskeleton and molecular motor proteins are known to play a pivotal role in most intracellular active transport processes. The actin depolymerizer cytochalasin D and microtubule effectors colchicine and paclitaxel were used to evaluate the function of these components of the cytoskeleton in the trafficking of polyethylenimine (PEI)-DNA complexes. In addition, ATPase inhibitors erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), vanadate, adenylylimidodiphosphate (AMP-PNP), and rose bengal lactone (RBL), which have inhibitory activity against dynein and kinesin, were used to examine to the effects of these molecular motors on PEI-DNA delivery. Disruption of microfilaments decreased the delivery efficiency of PEI polyplexes 60-80%, though cytochalasin D 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. Treatment with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by RBL (80%) and AMP-PNP (98%). Our findings confirm the importance of microfilament- and microtubule-based active transport of PEI-DNA complexes. Further, the strong decrease in transfection efficiency caused by ATPase inhibitors that possess inhibitory activity against kinesin implies an unexpected role for these motors in gene delivery.
Drake
David M
DM
Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, Illinois 61801, USA.
Pack
Daniel W
DW
D000199:Actins / Q000378:metabolism; D001693:Biological Transport, Active; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D004398:Dyneins / Q000037:antagonists & inhibitors; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors; D008870:Microtubules / Q000502:physiology; D011094:Polyethyleneimine / Q000493:pharmacokinetics*
D000199:Actins; D015638:Cytochalasin D; D011094:Polyethyleneimine; D004398:Dyneins; D016547:Kinesins