量子点（quantum dot）是纳米大小的发光晶体，可以从金、银等金属或者半导体中获得。由于有纳米级别的大小，量子点具有引人注目的光学性质：荧光强、稳定耐光、可激发多种荧光、激发光谱广和发射荧光光谱窄等优点。而且，由于比一般的荧光标记物质要小得多，所以不会干扰被标记分子的生物活性。正是因为如此，一直都是科学家荧光标记的重要候选物。不过，遗憾的是，量子点也有“先天不足”：生理环境会损害量子点的光学特性，反过来，量子点对机体核细胞也有毒。
虽然研究者苦苦探索，却一直都没有实现利用量子点进行活体定位标记和成像。
在最新的一期《Nature biotechnology》上，Emory University的科学家利用特别的生物兼容性量子点平台实现了这个长时间来梦寐以求的愿望。研究者利用一种ABC triblock共聚物包被由半导体硒化镉（cadmium selenide）产生的量子点，并进一步用聚乙烯包被。这种液态包被环境既能保存其光学特性，又能够保护不受外界极度 的酸碱盐条件的影响。
接下来是功能性试验中，研究者将这种包被的量子点连接到前列腺肿瘤特异的抗体上，并注入到前列腺肿瘤的小鼠，观察证明它们定位于前列腺肿瘤细胞。当小鼠被光照，这些量子点能够发光，表明肿瘤的部位和大小。
这项研究似乎揭示了量子点诊断的无限潜力，我们能够进一步研究，似乎可以预期这样的一种美好的前景：当病人接受一种追踪试剂，然后进行全身扫描，就可以知道潜在的肿瘤状况。然后，放大肿瘤部位，甚至可以了解细胞或者亚细胞水平的状况。
In vivo cancer targeting and imaging with semiconductor quantum dots
We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.
Figure 1. Schematic illustration of biconjugated QDs for in vivo cancer targeting and imaging.
(a) Structure of a multifunctional QD probe, showing the capping ligand TOPO, an encapsulating copolymer layer, tumor-targeting ligands (such as peptides, antibodies or small-molecule inhibitors) and polyethylene glycol (PEG). (b) Chemical modification of a triblock copolymer with an 8-carbon side chain. This hydrophobic side chain is directly attached to the hydrophilic acrylic acid segment and interacts strongly with the hydrophobic tails of TOPO. Dynamic light scattering shows a compact QD-polymer structure, indicating that QDs are tightly wrapped by the hydrophobic segments and hydrocarbon side chains. (c) Permeation and retention of QD probes via leaky tumor vasculatures (passive targeting) and high affinity binding of QD-antibody conjugates to tumor antigens (active targeting).
Figure 4. Spectral imaging of QD-PSMA Ab conjugates in live animals harboring C4-2 tumor xenografts.
Orange-red fluorescence signals indicate a prostate tumor growing in a live mouse (right). Control studies using a healthy mouse (no tumor) and the same amount of QD injection showed no localized fluorescence signals (left). (a) Original image; (b) unmixed autofluorescence image; (c) unmixed QD image; and (d) super-imposed image. After in vivo imaging, histological and immunocytochemical examinations confirmed that the QD signals came from an underlying tumor. Note that QDs in deep organs such as liver and spleen were not detected because of the limited penetration depth of visible light.
Figure 5. In vivo fluorescence images of tumor-bearing mice using QD probes with three different surface modifications: carboxylic acid groups (left), PEG groups (middle) and PEG-PSMA Ab conjugates (right).
For each surface modification, a color image (top), two fluorescence spectra from QD and animal skin (middle) and a spectrally resolved image (bottom) were obtained from the live mouse models bearing C4-2 human prostate tumors of similar sizes (0.5–1.0 cm in diameter). The amounts of injected QDs and the lengths of circulation were: 6 nmol and 6 h for the COOH probe; 6 nmol and 24 h for the PEG probe; and 0.4 nmol and 2 h for the PSMA probe (same as in Fig. 4). The site of QD injection was observed as a red spot on the mouse tail. The spectral feature at 700 nm (red curve, middle panel) was an artifact caused by mathematical fitting of the original QD spectrum, which has little or no effect on background removal.
Figure 6. Sensitivity and multicolor capability of QD imaging in live animals.
(a,b) Sensitivity and spectral comparison between QD-tagged and GFP-transfected cancer cells (a), and simultaneous in vivo imaging of multicolor QD-encoded microbeads (b). The right-hand images in a show QD-tagged cancer cells (orange, upper) and GFP-labeled cells (green, lower). Approximately 1,000 of the QD-labeled cells were injected on the right flank of a mouse, while the same number of GFP-labeled cells was injected on the left flank (circle) of the same animal. Similarly, the right-hand images in b show QD-encoded microbeads (0.5 m diameter) emitting green, yellow or red light. Approximately 1–2 million beads in each color were injected subcutaneously at three adjacent locations on a host animal. In both a and b, cell and animal imaging data were acquired with tungsten or mercury lamp excitation, a filter set designed for GFP fluorescence and true color digital cameras. Transfected cancer cell lines for high level expression of GFP were developed by using retroviral vectors, but the exact copy numbers of GFP per cell have not been determined quantitatively56.
