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Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells

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Abstract

Purpose

siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency.

Methods

Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers.

Results

Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process.

Conclusions

MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.

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Abbreviations

ADE:

ARF6-dependent endocytosis

ARF6:

ADP ribosylation factor 6

CME:

Clathrin-mediated endocytosis

CvME:

Caveolin-mediated endocytosis

DOPE:

Dioleoyl phosphatidylethanolamine

DOTAP:

N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate

FME:

Flotillin-mediated endocytosis

MP:

Macropinocytosis

mβCD:

Methyl-β-cyclodextrin

PEI:

Polyethylenimine

RISC:

RNA-induced silencing complex

RNAi:

RNA interference

siRNA:

Small interfering RNA

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Sandy Mattick at the Cell Culture Media Facility at the University of Illinois for help with preparation of cell media. We also thank Maureen K. Thompson for assistance with cell culturing and flow cytometry experiments, Dr. B. Pilas for help with flow cytometry and Dr. M. Sivaguru for the help with microscopy.

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Author notes

  1. Mihael Lazebnik

    Present address: Department of Early Stage Cell Culture, Genentech, Inc., South San Francisco, California, USA

  2. Rahul K. Keswani

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

Authors and Affiliations

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

    Mihael Lazebnik & Rahul K. Keswani

  2. Department of Chemical & Materials Engineering, University of Kentucky, Lexington, Kentucky, USA

    Daniel W. Pack

  3. Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, USA

    Daniel W. Pack

  4. University of Kentucky, 789 South Limestone, Lexington, Kentucky, 40536-0596, USA

    Daniel W. Pack

Authors
  1. Mihael Lazebnik
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Correspondence to Daniel W. Pack.

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Lazebnik, M., Keswani, R.K. & Pack, D.W. Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells. Pharm Res 33, 2999–3011 (2016). https://doi.org/10.1007/s11095-016-2022-1

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  • DOI: https://doi.org/10.1007/s11095-016-2022-1

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