Intracellular And In Vivo Applications

Quantum Dots. Latex beads (Meier et al., 2001) and dye molecules have long been used for tracking cell surface receptor dynamics. However, large nanoparticles can overwhelm the native physiology of the cell, and smaller dye molecules suffer from photobleaching. Quantum dots fall in the mesoscopic size range and do not suffer from photobleacing. Realizing the benefits of these unique properties of QDs, Dahan et al. (2003) have used quantum dots to study the diffusion dynamics of glycine receptors on the neuronal membrane of living cells. Glycine is an inhibitory neurotransmitter found in the central nervous system (Vannier and Triller, 1997). GlyR, the receptor for glycine, is present in clusters in the postsynaptic face of a nerve terminal (Triller et al., 1985). Gephyrin is a scaffolding protein known to stabilize these clusters or microdomains via mictotubule attachment of the GlyR receptors (Kirsch et al., 1993). The movement of these receptors within the cell membrane is critical in understanding synapse plasticity (Choquet and Triller, 2003). Dahan et al. first

Figure 4.7. Single QDs for live cell imaging. (A) Red QDs bound to GlyR receptor on neuron cell surface. Microtubule associated protein protein-2 is in green. [Reprinted from Dahan et al. (2003), with permission of American Association for the Advancement of Science.] (B) EGF-QDs endocytosed into CHO cells in a time dependent fashion. GFP-EGFR is in green. [Reprinted from Lidke et al. (2004), with permission of Nature Publishing Group.] See insert for color representation of this figure.

Figure 4.7. Single QDs for live cell imaging. (A) Red QDs bound to GlyR receptor on neuron cell surface. Microtubule associated protein protein-2 is in green. [Reprinted from Dahan et al. (2003), with permission of American Association for the Advancement of Science.] (B) EGF-QDs endocytosed into CHO cells in a time dependent fashion. GFP-EGFR is in green. [Reprinted from Lidke et al. (2004), with permission of Nature Publishing Group.] See insert for color representation of this figure.

incubated spinal cultured neurons with primary antibodies against the GlyR receptor on the neuron surface. Next, biotinylated secondary antibody fragments were allowed to bind to the primary antibodies on the cell surface, followed by addition of streptavidin-coated quantum dots. Single QDs were imaged with an exposure of 75 ms for approximately 60 s (Figure 4.7a). Even at such short exposure time, a signal-to-noise ratio of about 50 was obtained, which allowed single QD tracking on the cell surface with a lateral resolution of 5 nm. It was found that the GlyR receptors were distributed in three distinct regions: synaptic, perisynaptic, and extrasynaptic. Since GlyR receptors are clustered in the synaptic region, lower QD diffusion rates were observed in the synaptic and perisynaptic regions. Furthermore, the diffusion coefficients observed in the extrasynaptic region with the QDs were much higher than those observed with 500-nm latex in a previous study (Meier et al., 2001), confirming that large latex beads retarded the receptor diffusion in the membrane of the cell. Since QDs are electron-dense, the authors were also able to perform electron microscopy to confirm their findings. Following this report, Lidke et al. (2004) demonstrated use of QDs in probing the mechanism of erbB/HER receptor-mediated signal transduction. Briefly, CHO cells were incubated with streptavidin-coated QDs coupled with biotinylated epidermal growth factor (EGF). Binding of QD-EGF with the EGF receptor on the cell surface allowed observation of single QDs on the cell membrane and inside the cell after endocytosis (Figure 4.8b). Using the data obtained, the authors detected homodimer formation of the epidermal growth factor receptors on the cell surface.

For in vivo studies using animal models, Akerman et al. (2002) linked QDs with peptide ligands that were specific for murine lung cells. After injection of the QD-peptide construct through the tail vein of a mouse, the animal was sacrificed and its tissue was examined. The QDs were found to localize specifically in the lungs. This result was reinforced by similar experiments targeting blood and lymphatic vessels in tumors. Furthermore, PEG molecules (MW = 5000) on the surface improved circulation time and enabled the QD probe to evade the RES (reticuloendothelial system), which is the body's foreign particle filtration system. One step further, Dubertret et al. (2002) developed a more robust coating and demonstrated the biocompatibility and lack of toxicity for QD probes under in vivo conditions. A phospholipids-PEG block copolymer coating was used to stabilize the dots in an aqueous environment and render them biocompatible. The coated QDs were then injected into Xenopus embryos, which were used due to their sensitivity to toxicity and because small cellular disturbances could be revealed in biological phenotypes. Surprisingly, even when injected with 2 billion QDs per cell, the embryos showed little change in phenotype, although abnormal traits were evident at concentrations above 5 billion QDs per cell.

In a recent paper, Gao et al. (2004) reported in vivo cancer imaging and targeting with a new class of bioconjugated quantum dots. Antibodies specific to a prostate cancer marker called PSMA were conjugated to QDs coated with a PEGylated ABC triblock copolymer. The probes were injected into nude mice implanted with human prostate cancer cells and in vivo imaging was performed. The results showed QDs accumulated specifically at the implanted tumor site as shown in Figure 4.8. Due to high autofluorescence from the mouse skin, a spectral unmixing algorithm was applied to subtract the background fluorescence. This would not be necessary if NIR QDs are used because autofluorescence emission has been shown to taper off at ~800 nm and NIR spectral region lies in the range 700-900 nm. To that effect, another paper reported the use of NIR QDs for sentinel lymph node mapping (Kim et al., 2004). By injecting NIR QDs into pigs and mice and then imaging with an NIR imaging setup, a surgeon was able to identify the lymph node without interference from autofluorescence. Although this paper marks the first in vivo application of NIR QDs

Mouse QD-COOH

Mouse QD-PEG

Mouse QD-PSMA

True-color fluorescent images

Spectral unmixing

Mouse QD-COOH

Mouse QD-PEG

Mouse QD-PSMA

True-color fluorescent images

Spectral unmixing

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