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Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways

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Abstract

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 at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were 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 caveolin-dependent 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.

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Abbreviations

PEI:

Polyethylenimine

PAMAM:

Polyamidoamine

CTxB:

Cholera toxin subunit B

Tf:

Transferrin

DMEM:

Dulbecco’s modified Eagle’s medium

RLU:

Relative light units

MβCD:

Methyl-β-cyclodextrin

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ACKNOWLEDGMENTS AND DISCLOSURES

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.

Author information

Author notes

  1. Rahul K. Keswani

    Present address: Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, USA

Authors and Affiliations

  1. Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, Illinois, 61801, USA

    Mark E. Hwang & Daniel W. Pack

  2. Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, Urbana, Illinois, 61801, USA

    Rahul K. Keswani & Daniel W. Pack

  3. Department of Chemical and Materials Engineering and Department of Pharmaceutical Sciences, University of Kentucky, 467 Biological Pharmaceutical Building, 789 S. Limestone St., Lexington, Kentucky, 40536-0596, USA

    Daniel W. Pack

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  1. Mark E. Hwang
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Correspondence to Daniel W. Pack.

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Hwang, M.E., Keswani, R.K. & Pack, D.W. Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways. Pharm Res 32, 2051–2059 (2015). https://doi.org/10.1007/s11095-014-1598-6

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  • DOI: https://doi.org/10.1007/s11095-014-1598-6

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