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The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls
Dries Vercauteren1, Roosmarijn E Vandenbroucke, Arwyn T Jones
1Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Ghent, Belgium.
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-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD 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.
Insights
Common endocytosis inhibitors like chlorpromazine and MbetaCD can harm cell viability and lack specificity for distinct cellular uptake pathways. Their effects are also highly cell-dependent, questioning their use in gene delivery research without strict controls.
Area of Science:
- Cell Biology
- Biotechnology
- Pharmacology
Background:
- Nonviral gene complexes utilize various endocytic pathways for cellular entry.
- Understanding cellular uptake mechanisms is crucial for optimizing gene carrier efficiency.
- Current research often relies on chemical endocytosis inhibitors, but their reliability is debated.
Purpose of the Study:
- To characterize the effects of four common endocytosis inhibitors on cell viability and endocytic pathways.
- To assess the specificity and cell-line dependency of these inhibitors.
- To evaluate the suitability of these agents for studying gene carrier uptake mechanisms.
Main Methods:
- Tested chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion on five cell lines.
- Assessed cell viability following inhibitor treatment.
- Analyzed uptake and distribution of fluorescent probes (transferrin and lactosylceramide) for clathrin-dependent and -independent pathways.
Main Results:
- Chlorpromazine and MbetaCD reduced cell viability in some cell lines at standard inhibitory concentrations.
- Inhibitors showed poor specificity in distinguishing between clathrin-dependent and -independent endocytosis.
- Observed significant cell-line-dependent variations in inhibitor effects.
Conclusions:
- The study questions the reliability of commonly used endocytosis inhibitors due to their impact on cell viability and lack of pathway specificity.
- Findings highlight the critical need for stringent controls when using these agents in endocytosis research.
- Results suggest that alternative methods may be necessary for accurate profiling of gene carrier cellular uptake.
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