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.
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