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Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells
Eva M Luther1, Charlotte Petters, Felix Bulcke
1Center for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany.
Abstract:
Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ∼65 nm and -49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ∼130 nm and -8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3h with 150 and 450 μM iron as BP-IONPs increased the cellular iron content from a low basal level of ∼50 nmol iron mg(-1) to 219±52 and 481±28 nmol iron (mg protein)(-1), respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.
Insights
Iron oxide nanoparticles (IONPs) are rapidly taken up by microglial cells, the brain's immune cells. This uptake occurs via macropinocytosis and clathrin-mediated endocytosis, directing IONPs to lysosomes.
Area of Science:
- Neuroscience
- Nanotechnology
- Cell Biology
Background:
- Microglia are crucial brain phagocytes responding to homeostasis changes.
- Iron oxide nanoparticles (IONPs) have emerging diagnostic and therapeutic uses in the brain.
- Understanding microglial interaction with IONPs is vital for neuro-applications.
Purpose of the Study:
- To synthesize and characterize fluorescent BODIPY®-labelled IONPs (BP-IONPs).
- To investigate the uptake mechanisms and cellular fate of BP-IONPs in microglial cells.
- To assess the impact of BP-IONPs on microglial cell viability.
Main Methods:
- Synthesis and characterization of fluorescently labelled IONPs (BP-IONPs).
- Exposure of cultured rat microglial cells to varying concentrations and durations of BP-IONPs.
- Measurement of cellular iron content and fluorescence.
- Viability assays and co-localization studies with lysosomal markers.
- Inhibition studies using specific endocytosis pathway blockers.
Main Results:
- BP-IONPs were successfully synthesized and characterized, with size and surface charge varying in different media.
- Microglial cells exhibited time-, concentration-, and temperature-dependent uptake of BP-IONPs.
- Cell viability was maintained at lower concentrations/shorter durations but compromised at higher levels or longer exposures.
- BP-IONP fluorescence localized to lysosomes within microglial cells.
- Uptake was significantly reduced by inhibitors of macropinocytosis and clathrin-mediated endocytosis.
Conclusions:
- Microglial cells rapidly internalize BP-IONPs.
- The primary uptake pathways are macropinocytosis and clathrin-mediated endocytosis.
- BP-IONPs are directed to the lysosomal compartment after cellular uptake.
- These findings are critical for the safe application of IONPs in neuro-diagnostics and therapeutics.
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