Purpose Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. Methods We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes atN/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitorswere used at varying concentrations to elucidate the roles of these important pathways. Results PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolindependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. Conclusions The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
|Number of pages||9|
|State||Published - Jun 1 2015|
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health grants GM085222 and DK083875 (to M.E.H.). We thank Sandy McMasters at the Cell Media Facility and Barbara Pilas at the Flow Cytometry facility at the University of Illinois. The authors have no competing financial interests relating to the work reported.
© Springer Science+Business Media New York 2014.
- Polymer gene delivery
ASJC Scopus subject areas
- Molecular Medicine
- Pharmaceutical Science
- Organic Chemistry
- Pharmacology (medical)