The use of inhibitors to study endocytic pathways of gene carriers: Optimization and pitfalls

Vercauteren, Dries, Vandenbroucke, Roosmarijn E., Jones, Arwyn Tomos ORCID: https://orcid.org/0000-0003-2781-8905, Rejman, Joanna, Demeester, Joseph, De Smedt, Stefaan C., Sanders, Niek N. and Braeckmans, Kevin 2010. The use of inhibitors to study endocytic pathways of gene carriers: Optimization and pitfalls. Molecular Therapy 18 (3) , pp. 561-569. 10.1038/mt.2009.281

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Abstract

Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-β-cyclodextrin (MβCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MβCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Publisher:	Elsevier
ISSN:	1525-0016
Last Modified:	17 Oct 2022 10:05
URI:	https://orca.cardiff.ac.uk/id/eprint/6753

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