IN VIVO CELL TARGETING / IMAGING WITH NEAR-IR-EMITTING DOPED ZNSE QUANTUM DOTS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 量子点(QD)由于其独特的光学性质，如强烈的荧光信号、高抗光漂白和宽的激发光谱，允许在单一激发源下的多路应用，在各种生物研究中作为体内荧光团受到了极大的关注。到目前为止，研究最广泛的量子点是很容易从商业来源获得的是镉、镉和铅。然而，镉和铅的固有毒性使其在体内长期研究中的适用性受到质疑。针对这一问题，我们开发了一种新的绿色化学合成路线，在水环境中制备了一系列无镉、无铅的量子点。体外细胞活性测试表明，谷胱甘肽(GSH)稳定的量子点毒性较低。此外，这些量子点的发射在红色到近红外(650-800 nm)的范围内是可调节的，这与生物的传输窗口相吻合，为细胞和小动物的荧光成像提供了高信噪比。最重要的是，这些高度水溶性的、生物相容的量子点很容易通过与GSH上的羧基/氨基连接的生物分子或包裹在生物聚合物中的生物分子与生物系统连接。 在拟议的研究中，我们将探索合成的量子点在体内长期成像、跟踪和靶向方面的应用。我们将在以下具体目标的框架内这样做： 目的：研究生物聚合物/靶标配体修饰的量子点在体内的短期生物分布、清除及潜在毒性。量子点的生物分布动力学是其对靶组织的特定作用以及在全身应用后识别任何不良副作用的重要方面。在这项研究中，量子点将被注射到裸鼠的尾静脉中，并在注射后的不同时间点使用荧光成像系统进行成像。小鼠将在研究接近尾声时被处死，它们的主要器官将通过手术暴露并进行成像。 目的2：监测肥大细胞来源的颗粒向引流淋巴结(DLN)的转运途径。在感染期间，信号分子可以穿越很长的距离到达DLN。然而，目前还不清楚这些分子如何在不被稀释或降解的情况下通过这条路线。肥大细胞一旦被激活，就会释放含有肿瘤坏死因子和其他蛋白质的稳定的基于肝素的颗粒。我们建议用我们的量子点标记这些MC部分，注射到MC缺陷的KitW-sh/W-sh小鼠/C57BL/6小鼠的足垫中，用荧光成像系统观察其运输过程2小时，实验结束后处死小鼠，对其主要器官进行组织学分析。(本研究也适用于监测经口服或静脉注射进入SD大鼠体内的量子点标记纳米粒的情况。在这种情况下，将使用荧光成像系统对大鼠进行为期1周的监测，在研究结束时处死大鼠，并对其主要器官进行手术曝光和成像)。 目的3：对量子点标记的干细胞进行体内迁移/分化的长期跟踪。干细胞的潜在应用之一是用于再生医学；然而，对于临床前和临床试验，重要的是拥有一种非侵入性技术来评估移植的干细胞的治疗效果和位置，以排除潜在的副作用。在这个实验中，我们将结合特定的量子点抗体，这些抗体将靶向人类间充质干细胞(HMSCs)的表面标记。QD标记的hMSCs将被静脉注射到NOD/SCID小鼠体内，并将在8周内使用荧光成像系统对小鼠进行监测，以了解干细胞的命运。 目的4：研制可用于体内肿瘤诊断的高灵敏度量子点探针。癌症诊断的成功与否与发现恶性肿瘤的阶段有关。然而，目前能发现早期癌症的敏感测试很少。为了进一步说明量子点作为体内靶向和跟踪能力的广泛应用，我们建议进行这项研究，首先将我们的水量子点与活性的肿瘤靶向配体/治疗剂结合在一起，以结合到早期深层组织肿瘤上，并通过荧光成像建立对药物动力学和疾病治疗的实时监测。体内靶向/跟踪研究将在裸鼠身上进行，为期3个月。实验接近尾声时，处死小鼠，进行组织学检查。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Quantum dots (QDs) are appealing as in vivo fluorophores in a variety of biological investigation due to their unique optical properties such as strong fluorescence signal, high resistance to photobleaching, and broad excitation spectra that allows multiplexing application with a single excitation source. To date, the most widely studied QDs that are readily available from commercial sources are CdSe, CdTe and PbSe. However, the intrinsic toxicity of cadmium and lead sheds a doubt on its applicability for long term in vivo studies. In view of this issue, we have developed a novel green chemistry synthetic route to produce a series of cadmium and lead free QDs in aqueous environment. The as-synthesized QDs that are stabilized with glutathione (GSH) exhibit low toxicity as demonstrated by in vitro cell viability test. In addition, the emission of these QDs are tuanable in the red to near-infrared range (650-800 nm), that coincide with the biological window of transmission to offer high signal-to-noise for fluorescence imaging of cells and small animals. Above all, these highly water-soluble, biocompatible QDs are readily amendable to interfacing with biological systems through biomolecules conjugated with the carboxyl/amino groups on GSH or encapsulated in biopolymers. In the proposed study, we will explore the applications of the as-synthesized QDs for long-term in vivo imaging, tracking and targeting. We will do so within the framework of the following specific aims: Aim 1: To study the short-term in vivo biodistribution, clearance, and potential toxicity of the as-synthesized QDs/QDs encapsulated in biopolymers/QDs conjugated with targating ligands over a period of 1 week. Biodistribution dynamics of QDs is a vital aspect for their specific effects on target tissue as well as to identify any undesired side effects after systemic application. In this study, QDs will be injected into the tail vein of nude mice and imaged at various time points post-injection using a fluorescent imaging system. The mice will be sacrificed towards the end of the study and their major organs will be surgically exposed and imaged. Aim 2: To monitor the route of trafficking of mast cell-derived particles to draining lymph nodes (DLNs). During infection, signalling molecules could traverse significant distances to reach the DLNs. However, it is unclear how these molecules could go through the route without dilution or degradation. Mast cells, upon activation, have shown to release stable heparin-based particles containing tumor necrosis factor and other proteins. We propose to label these MC partlces with our QDs, inject into the footpads of MC deficient KitW-sh/W-sh mice / C57BL/6 mice and visualize the trafficking process using the fluorescence imaging system for 2 h. The mice will be sacrificed at the end of the experiment and histological anaylsis will be done on their major organs. (This study is also applicable for monitoring the of QDs labeled nanoparticles introduced into Sprague Dawley rat through oral route or intravenous injection. For this case, the rat will be monitored for 1 week using the fluorescence imaging system, sacrificed at the end of the study and their major organs will be surgically exposed and imaged). Aim 3: To perform long-term in vivo tracking of the migration/differentiation of QDs-labeled stem cells. One of the potential applications of stem cells is for regenerative medicine; however for pre-clinical and clinical trials, it is important to have a noninvasive technique to evaluate the therapeutic effect and location of the implanted stem cells to rule out potential side effects. For this experoment we will conjugate specific antibodies to QDs that will target the surface markers on human mesenchymal stem cells (hMSCs). The QD-labeled hMSCs will be injected intravenously in NOD/SCID mice and the mice will be monitored using the fluorescent imaging system over a period of 8 weeks to access the fate of the stem cells. Aim 4: To develop QDs as highly sensitive probes for in vivo cancer diagnosis. The success of cancer diagnosis is related to the stage at which the malignancy is detected. However, at present there are very few sensitive tests that could detect early-stage cancers. To further illustrate the vast applications of QDs as in vivo targeting and tracking capabilities, we propose to conduct this study by first conjugate our aqueous QDs with active tumor targeting ligands/therapeutic agents to bind to early-stage deep tissue tumor and to establish real-time monitoring of pharmacokinetics and disease treatment through fluorescence imaging. In vivo targeting/tracking studies would be carried out on nude mice grown with human prostate cancer for a period of 3 months. Towards the end of the study, the mice would be sacrificed and histological examination would be carried out.