Our study shows that QDs are preferentially taken up by microglia in mixed cortical cultures and in brain. We further showed that the major cellular uptake pathway of QDs in microglia is clathrin-mediated endocytosis involving the microglia-specific receptors MSR-1 and mannose receptor. In primary mixed cortical cultures, QDs effectively delivered the cytotoxin saporin selectively to microglia. Depletion of microglia with QD-Sap resulted in protection against microglia-mediated Aβ toxicity.
Our finding that QDs are selectively taken up by microglia is consistent with previous observations that QDs were localized to macrophages and microglia that infiltrate experimental gliomas . However, in contrast to the previous study, which suggested that QDs were phagocytosed by macrophages and microglia, our data indicate that QDs enter microglia via receptor binding and clathrin-mediated endocytosis. In eukaryotes, macromolecules enter the cell in membrane-bound vesicles either via 'phagocytosis' (the uptake of particles larger than 0.5 μm in diameter) or 'pinocytosis' (the uptake of fluid and solutes) . Phagocytosis occurs by an actin-dependent mechanism and is usually independent of pH gradient and clathrin, whereas pinocytosis occurs by at least four basic mechanisms: macropinocytosis, clathrin-mediated endocytosis, calveolae-mediated endocytosis, and clathrin- and calveolae-independent endocytosis . Interestingly, the uptake of soluble Aβ by microglia was found to be mediated through a nonsaturable, fluid phase macropinocytic mechanism that is distinct from phagocytosis and receptor-mediated endocytosis . The size of QDs, which range from 10-100 nm in diameter, makes it unlikely that QDs enter microglia via phagocytosis. Indeed, the blockade of QD entry by balifomycin, chlorpromazine, and cytochalasin B provide strong evidence that QDs are taken up by microglia via clathrin-mediated endocytosis. Clathrin-mediated endocytosis occurs in all cell types. However, blocking the MSR-1 or mannose receptor with specific inhibitors or antibodies prevented the uptake of QDs, indicating that the selective targeting of QDs to microglia requires their binding to microglia-specific receptors. We cannot exclude that other microglial receptors such as Fc-receptors, complement receptors, and Toll-like receptors might also mediate the endocytosis of QDs by microglia. Additional studies are needed to fully characterize the potential binding sites of QDs on the microglial surface.
The unique optical properties of quantum dots, such as high quantum yields, large molar extinction coefficients, size-dependent tunable emission and high photostability, make them appealing as fluorescent probes for biological imaging. On the other hand, because of their size range, QDs are also very suitable for manipulations at the molecular level, offering new approaches for the delivery of potent bioactive agents. Microglia may have roles in the pathogenesis of various CNS diseases, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, and amyotropic lateral sclerosis [36–39]. Our finding that certain sizes of QDs selectively target microglia provides a novel platform to probe and modulate biological processes in microglia and may lay the foundation for the development of QD-based reagents that can modulate specific signaling pathways in microglia.
In contemplating the therapeutic potential of QDs, an important caveat is their biocompatibility and toxicity [18, 40]. The use of PEG on the surface of the QDs significantly improved their biocompatibility and minimized their toxicity [41–43]. A gene profiling study showed that application of high-dose QDs only induced changes in a small number of genes associated with the transport machinery, supporting the feasibility of long-term usage of QDs in biological systems . The current study provides evidence that targeting of microglia with QDs is unlikely to result in toxicity through increased cytokine release, even in the presence of LPS-stimulated microglial activation. Indeed, our data suggest that the toxicity of QDs is limited, at least in the short term. However, evidence suggests that QDs may activate autophagy, implicating an important role in the regulation of normal cell processes [44–47]. The size-dependent induction of autophagy by QDs could result in the initiation of a cell death cascade . Alternatively, the induction of autophagy during inflammation may protect against the harmful effects of microglial activation. Further investigation will be required to establish the long-term effects of the material, especially the heavy-metal component, in biological systems. If their safety profile continues to improve, QDs may emerge as a novel approach for the selective delivery of therapeutic agents to microglia in diverse CNS diseases.