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WIREs Nanomed Nanobiotechnol
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Intracellular imaging of quantum dots, gold, and iron oxide nanoparticles with associated endocytic pathways

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Metallic nanoparticles (NP) have been used for biomedical applications especially for imaging. Compared to nonmetallic NP, metallic NP provide high contrast images because of their optical light scattering, magnetic resonance, X‐ray absorption, or other physicochemical properties. In this review, a series of in vitro imaging techniques for metallic NP will be introduced, meanwhile their strengths and weaknesses will be discussed. By utilizing these imaging methods, the cellular uptake of metallic NP can be easily visualized to better understand the endocytic mechanisms of NP intracellular delivery. Several types of metallic NP that are used for imaging or as contrast agents such as quantum dots, gold, iron oxide, and other metallic NP will be presented. Cellular uptake of metallic NP and associated endocytic mechanisms highly depends upon the NP size, charge, surface coating, shape, or other factors such as cell type, cell differentiation status, cell surface status, external forces, protein binding, temperature, and the biological milieu. Classical endocytic routes such as lipid raft‐mediated pathways, clathrin or caveolae‐mediated pathways, macropinocytosis, and phagocytosis have been investigated, yet there is still a demand to determine other endocytic pathways. Knowing the different methodologies used to determine the endocytic pathways will increase the understanding of NP toxicity, cancer cell targeting, and imaging, so that surface coatings can be created for efficient cell uptake of metallic NP with minimal cytotoxicity WIREs Nanomed Nanobiotechnol 2017, 9:e1419. doi: 10.1002/wnan.1419 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
QD tracking on the surface of rat basophilic leukemia (RBL) cells by TIRF imaging. QD were incubated with RBL for over 46 min and excited with emission wavelength of 605 nm. TIRF images were recorded with intervals at 3 s to visualize the QD spot internalization in the cells. (Reprinted with permission from Ref . Copyright 2011 Wiley)
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AuNP endocytic stages. (a) Chemotaxis forming a vauole using its pseudopodia; (b) phagocytosis through formation of phagolysosome (PL) upon fusion with lysosomes (L); (c) digestion: peroxisome (Pe) and lysosome (L) fuse with phagosome (Ph) forming a phagolysosome (PL). (Reprinted with permission from Ref . Copyright 2010 American Chemical Society)
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QD655‐COOH cellular uptake in DC imaged by fluorescence microscopy. QD655‐COOH at 0.05nM were incubated with DC for 30 min. Fluorescent images were captured in the differential interference contrast (DIC) mode and QD channel. (Reprinted with permission from Ref . Copyright 2011 Future Medicine)
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QD endocytic uptake mechanism and subcellular localization with 24 inhibitors with different inhibitory functions and protein markers for organelles. Lipid rafts, caveolin1, clathrin, early endosome, and late lysosomes are marked with orange color. Inhibitors are located near the targets where they exert their functions. Inhibitors with effects are labeled in green while inhibitors without effects are labeled in black. Briefly, QD were first recognized by lipid rafts (CTX) and was internalized into early endosome (EEA1), then localized in late endosome (CD63) and remained in the lysosome (LAMP1). QD uptake was through theG protein‐coupled receptor (GPCR) and the downstream proteins regulated by Giprotein, PLC, and PKC. These inhibitors blocked QD uptake, indicating QD endocytosis may be recognized by specific receptors. The inhibitors for scavenger receptors polyI and fuicodan (FCD) greatly blocked QD. LDL/AcLDL competed with QD and reduced QD internalization suggesting that LDLR/SR‐BI may be the most appropriate receptors of QD uptake.Other abbreviations for this figure: lysosome‐associated membrane glycoproteins1 (LAMP1), cholera toxin B (CTB), low density lipoprotein (LDL), Acetylated LDL (AcLDL), cytochalasin D (CytD), methyl‐b‐cyclodextrin (MβCD), 5‐(N,N‐dimethyl)‐amiloride (DMA),sodium azide (NaN3), wortmannin (WMN), Ly294002 (LY), pertussis toxin(PTX), cholera toxin (CTX), staurosporine (SRP), bafilomycin A1 (BMA1), chloroquine (CRQ), trypsin inhibitor (TrpI), niacinamide (NCM), chlorpromazine (CPM), brefeldin A (BFA).
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QD colocalization with EEA1 and LAMP1. EEA1 staining (green) in HEK with QD655‐COOH at 1 h showed colocalization of QD with early endosomes. At 12 h, LAMP1 staining of HEK showed the colocalization of QD with lysosomes. Note QD distribution in HEK shows a punctate pattern at 1 h and a perinuclear pattern at 12 h.
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Intracellular imaging of AuNP. (a) AuNP imaging by dark field microscopy. AuNP at 50 nm in TEM size were incubated with HepG2 cells for 24 h. Dark field microscopy was used to image the reflectance of AuNP, while the nuclei was stained with 4',6‐diamidino‐2‐phenylindole (DAPI). (b) AuNP imaging in the skin tissue by multiphoton microscopy. Blue‐green color indicates the autofluorescence attributed from endogenous NAD(P)H, while AuNP showed an orange to red color. (Reprinted with permission from Ref . Copyright 2011 Springer)
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