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. 2012 Jan 24:9:22.
doi: 10.1186/1742-2094-9-22.

Selective targeting of microglia by quantum dots

S Sakura Minami  1 , Binggui Sun, Ketul Popat, Tiina Kauppinen, Mike Pleiss, Yungui Zhou, Michael E Ward, Paul Floreancig, Lennart Mucke, Tejal Desai, Li Gan
Affiliations

Selective targeting of microglia by quantum dots

S Sakura Minami et al. J Neuroinflammation. .

Abstract

Background: Microglia, the resident immune cells of the brain, have been implicated in brain injury and various neurological disorders. However, their precise roles in different pathophysiological situations remain enigmatic and may range from detrimental to protective. Targeting the delivery of biologically active compounds to microglia could help elucidate these roles and facilitate the therapeutic modulation of microglial functions in neurological diseases.

Methods: Here we employ primary cell cultures and stereotaxic injections into mouse brain to investigate the cell type specific localization of semiconductor quantum dots (QDs) in vitro and in vivo. Two potential receptors for QDs are identified using pharmacological inhibitors and neutralizing antibodies.

Results: In mixed primary cortical cultures, QDs were selectively taken up by microglia; this uptake was decreased by inhibitors of clathrin-dependent endocytosis, implicating the endosomal pathway as the major route of entry for QDs into microglia. Furthermore, inhibiting mannose receptors and macrophage scavenger receptors blocked the uptake of QDs by microglia, indicating that QD uptake occurs through microglia-specific receptor endocytosis. When injected into the brain, QDs were taken up primarily by microglia and with high efficiency. In primary cortical cultures, QDs conjugated to the toxin saporin depleted microglia in mixed primary cortical cultures, protecting neurons in these cultures against amyloid beta-induced neurotoxicity.

Conclusions: These findings demonstrate that QDs can be used to specifically label and modulate microglia in primary cortical cultures and in brain and may allow for the selective delivery of therapeutic agents to these cells.

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Figures

Figure 1
Figure 1
Selective uptake of QDs by microglia in mixed primary cortical cultures. A. In mixed primary cortical cultures from rats, QDs (red) were internalized primarily by microglia labeled with anti-CD11b (green) or anti-Iba-1 (green) antibodies. Nuclei were labeled with DAPI (blue). Scale bar, 50 μm. Inset, higher magnification of a QD-containing microglia cell labeled with Iba-1 antibody. B, C. QDs (red) were not found in astrocytes labeled with anti-GFAP (green, B) or in neurons labeled with anti-MAP2 (green, C). Scale bar, 50 μm. D. Fluorescence intensity of QDs in microglia increased with time of incubation. n = 30-100 cells from 5-6 independent images measured with Metamorph analyses. * P < 0.05 (24 h vs. 48 h), *** P < 0.001 (3 h vs. 8 h, and 8 h vs. 24 h); one-way ANOVA with Tukey-Kramer posthoc analyses. Error bars represent SEM. E. QDs (red) were not detected in apoptotic or necrotic cells labeled with Annexin V/Sytox green (green). Scale bar, 50 μm. F. Microglial internalization of QD655 conjugated with polyethylene glycol (QD655-PEG), PEG-streptavidin (QD655-PEG-Strep), or -COO- (QD655-Carboxyl) in mixed cortical cultures. Red: QDs; green: Iba-1-positive microglia. Scale bar, 50 μm.
Figure 2
Figure 2
Size of QDs affects their uptake by microglia. A. Internalization of QDs of different sizes by microglia. Blue: DAPI. B. Size-dependent internalization of QDs by microglia was quantified by fluorescence intensity with Metamorph analyses. n = 23-39 cells from 5-6 independent images. ***, P < 0.001 by Tukey-Kramer posthoc test. Bars represent mean ± SEM. Scale bar, 20 μm.
Figure 3
Figure 3
The uptake of QDs by microglia depends on clathrin-mediated endocytosis. The internalization of QDs by microglia was blocked dose-dependently by bafilomycin (BAF) (A, B), chlorpromazine (CPZ) (C, D), or cytochalasin B (CTB) (E, F). n = 18-48 cells per condition. ** P < 0.01, *** P < 0.001, vs. Vehicle (0.1% ethanol) or non-treated (NT) by Tukey-Kramer posthoc test. Bars represent mean ± SEM. Scale bar, 20 μm.
Figure 4
Figure 4
Microglial uptake of QDs occurs through mannose receptors and macrophage scavenger receptors. A. The internalization of QDs by microglia in rat primary mixed cortical cultures was quantified by fluorescence intensity with Metamorph software. Microglial uptake of QDs was blocked in a dose-dependent manner by mannan, an inhibitor of mannose receptors (A) and by polyinosinic acid (PIA), an inhibitor of scavenger receptors (B). n = 120-200 cells from at least eight separate images. Experiments were repeated three times. C. Antibodies against mannose receptor (MR) or macrophage scavenger receptor (MSR) were applied to rat primary mixed cortical cultures at a 2 μM concentration for 2 h before incubation with QDs. Microglial uptake of QDs was blocked by antibodies against MR and MSR. n = 105-120 cells from at least eight separate images. Experiments were repeated twice. ***P < 0.001, **P < 0.01, *P < 0.05 vs. control-treated, one-way ANOVA with Tukey-Kramer posthoc analyses. Bars represent mean ± SEM.
Figure 5
Figure 5
QDs are selectively taken up by microglia in vivo. A-D. QDs were stereotaxically injected into the hippocampus of CX3CR+/- mice, which express GFP in microglia. A. Representative photomicrographs showing the distribution of QDs in the hippocampus. B. GFP-labeled microglia in the hippocampus. C. Merge of (A) and (B) shows that the majority of QDs were localized in microglia. Scale bar, 200 μm. D. Higher magnification image of the boxed area in (C). Scale bar, 20 μm. E. Representative confocal image through a single plane of quantum dots (red) internalized by microglia (green) in the hippocampus of CX3CR+/- mice. Blue: DAPI. Scale bar, 20 μm. F, G. QDs (red) were not taken up by astrocytes labeled with anti-GFAP (green, F) or neurons labeled with anti-MAP2 (green, G) after they were injected into the hippocampus of C57Bl/6 wildtype mice. Scale bar, 20 μm.
Figure 6
Figure 6
Long-term expression of QDs. A, B. QDs are observed 7 days (A) and 28 days (B) after injection into the hippocampus of adult C57BL/6 mice. Scale bar, 200 μm.
Figure 7
Figure 7
Conjugation of QDs with saporin by streptavidin-biotin binding. A. Comparison of carbon composition of QDs and QD-saporin conjugates measured by X-ray photoelectron spectroscopy. B. High-resolution C1 scans confirmed the presence of saporin on the surface of QDs.
Figure 8
Figure 8
QD-saporin-mediated depletion of microglia decreases neuronal loss in mixed cortical cultures exposed to Aβ. A. Representative photomicrographs of microglial cells in mixed cortical cultures treated with QDs or QD-saporin (QD-Sap) conjugates. Microglial cells were labeled with anti-Iba-1 (green) and nuclei were labeled with DAPI (blue). B. Quantification of Iba-1-positive cells in mixed cultures treated with unconjugated saporin (Sap), QDs, or QD-saporin conjugates (QD-Sap). C. Quantification of MAP2-positive neurons in mixed cultures treated with Aβ1-42 after pretreatment with medium alone, QDs, or QD-Sap. n = 5 wells from three independent experiments. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey-Kramer posthoc analyses. Scale bar, 100 μm.
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