Lymphatic Imaging

淋巴管是人体的“第三循环”，是肿瘤扩散的重要途径。淋巴管生成是一些肿瘤的特性，允许它们通过淋巴管繁殖。这个过程很难研究，因为淋巴管很小，难以接近。然而，通过注射各种大分子造影剂，包括放射性标记、光学标记和磁性标记，可以识别淋巴管及其引流模式(1)。此外，在此过程中获得的知识对原发性和继发性淋巴水肿的诊断和管理具有重要意义，这可能是癌症治疗的严重并发症。最后，大分子造影剂的发展对其他疾病也有影响。临床前研究MIP正在使用成像方法研究淋巴管在癌症中的作用。我们发现直径在5-10nm之间的大分子药物是研究淋巴的理想药物，因为它们能快速吸收并转运到前哨淋巴结。前哨淋巴结成像很重要，因为目前的方法依赖于繁琐的蓝色染料和放射性硫胶体。我们已经开发出一种树突状分子，可用于MRI成像和光学成像的双重标记，可以在不使用MRI和光学成像电离辐射的情况下识别前哨淋巴结(2)。通过比较不同大小的树状大分子，实现了淋巴剂的优化(3)。我们发现非常小的药剂从淋巴中泄漏，效果不理想，而大的药剂则太慢。因此，第6代负载钆和光学荧光团的树状大分子是理想的（3-5）。与Martin Brechbiel的团队合作，我们开发并测试了新的树状大分子合成方法，这将简化化学反应，可能用于临床翻译(6)。我们还使用IgG作为淋巴成像的载体分子，并证明它具有生物相容性和适合淋巴成像的大小。此外，我们正在探索量子点（QD）在识别淋巴管中的作用。量子点具有量子效率高和发射光谱非常窄的优点，可以同时使用多个不同波长的量子点（7,8）。我们使用了两个彩色量子点来研究乳房和手臂向腋窝淋巴结的引流，发现一些小鼠具有独立的引流系统，而另一些小鼠的淋巴结具有“分水岭”淋巴结，即部分引流乳房和部分引流肢体的淋巴结（7,8）。有趣的是，该节点根据其排水情况被划分为单独的隔间。这种解剖对免疫抗原处理的意义是潜在的兴趣。此外，我们还探索了同时使用多达5个不同波长的量子点，以建立头部和颈部复杂的淋巴引流模式。这些研究表明，确定特定区域的流域和显示重叠流域是可能的。在过去的一年里，我们写了一篇关于淋巴成像主题的综述(1)。临床翻译：我们一直在与波士顿儿童医院合作开发一种基于树突的MR造影剂，用于患有原发性淋巴水肿的儿童。截至2007年9月，Brechbiel博士的团队已经合成了用于猪模型实验的材料，MIP人员将协助波士顿儿童医院进行这些研究。这项工作的一个重要副产品是我们已经生成了钆标记的白蛋白分子。每个白蛋白用10-15个钆原子标记。因此，它在MRI上是一种很好的显像剂，可以用作替代标记物。Drs。Ed Oldfield和Russ Lonser表示有兴趣将这种药物用作脑肿瘤患者对流增强输送（CED）药物的替代标记物。目前的CED治疗由于无法监测药物而受到限制。由于gd -白蛋白表现出与CED （IL-13 PEG）类似的对流特性，它将是一个有用的替代标记物。JDC批准了gd -白蛋白的GMP生产，用于临床中心的CED试验。GMP生产合同被授予肯塔基大学列克星敦分校，GMP合成正在进行中（2007年9月）。1. Barrett， T., Choyke， P. L.和Kobayashi， H.淋巴系统成像：新视野。中国生物医学工程学报，2006,31(2):557 - 557。2. Talanov， V. S, Regino， C. A, Kobayashi， H., Bernardo， M., Choyke， P. L.和Brechbiel， M. W.基于树突分子的纳米探针，用于双模态磁共振和荧光成像。生物工程学报，6(6):1459-1463,2006。3. Kobayashi， H., Kawamoto， S., Bernardo， M., Brechbiel， M. W., Knopp， M. V.，和Choyke， P. L.。向前哨淋巴结输送钆标记纳米粒子：前哨淋巴结可视化的比较和核磁共振成像对淋巴结内钆浓度的估计。[J] .中国机械工程，2009,31(2):444 - 444。4. Koyama， Y., Talanov， V. S., Bernardo， M., Hama， Y., Regino， C. A., Brechbiel， M. W., Choyke， P. L.和Kobayashi， H.一种树突状纳米造影剂，用于磁共振和光学荧光成像定位小鼠前哨淋巴结。中国生物医学工程学报，25(2):866-871,2007。5. Hama， Y., Bernardo， M., Regino， C. A., Koyama， Y., Brechbiel， M. W., Krishna， M. C., Choyke， P. L.和Kobayashi， H.使用树突状造影剂的MR淋巴管造影：1.5T和3.0T的比较。中华检验医学杂志，2009,31(4):431-436。6. Xu， H., Regino， C. A., Bernardo， M., Koyama， Y., Kobayashi， H., Choyke， P. L.，和Brechbiel， M. W.改进树突基磁共振成像造影剂的合成：新的双功能二乙烯三胺五乙酸配体和非水偶联化学。中华医学杂志，2009,35(3):385 - 393。7. Kobayashi， H., Hama， Y., Koyama， Y., Barrett， T., Regino， C. A., Urano， Y.，和Choyke， P. L.使用量子点对五个不同淋巴池同时进行多色成像。生物工程学报，7(7):1711-1716,2007。8. Hama， Y., Koyama， Y., Urano， Y., Choyke， P. L.和Kobayashi， H.使用ig共轭近红外光学探针的双色淋巴映射。[J]皮肤科杂志，2007。

Imaging of Lymphatics Background The lymphatics represent the "third circulation" in the body and are an important route of spread for tumors. Lymphangiogenesis is a property of some tumors that allows them to propagate via the lymphatics. This process has been difficult to study because the lymphatics are small and inaccessible. However, by injecting various kinds of macromolecular contrast agents, including radiolabeled, optically labeled and magnetically labeled it is possible to identify the lymphatics and their drainage patterns(1). Moreover, the knowledge gained in this process has implications for the diagnosis and management of primary and secondary lymphedema, which can be a serious complication of cancer therapy. Finally, the development of macromolecular contrast agents has implications for other diseases. Pre-clinical research The MIP is investigating the role of the lymphatics in cancer using imaging methods. We have found that macromolecular agents of between 5-10nm in diameter are ideal for studying the lymphatics based on their rapid uptake and transit to the sentinel nodes. Sentinel node imaging is of importance because current methods rely on cumbersome blue dyes and radioactive sulfur colloid. We have developed a dendrimer that is dual labeled for MRI imaging and optical imaging that would allow the identification of the sentinel node without ionizing radiation using MRI and optical imaging(2). By comparing different sized dendrimers optimization of the lymphatic agent was achieved(3). We found that very small agents leaked from the lymphatics and were unsatisfactory whereas large agents were too slow. Therefore, the Generation 6 dendrimer loaded with Gadolinium and an optical fluorophore appears ideal(3-5). Working with Martin Brechbiel's group we have developed and tested dendrimers with new syntheses that should simplify the chemistry for possible clinical translation(6). We have also used IgG as a carrier molecule for lymphatic imaging and shown it to be a biocompatible and suitably sized for lymphatic imaging. In addition we are exploring the role of quantum dots (QD) in identifying the lymphatics. QDs have the advantages of high quantum efficiency and very narrow emission spectra allowing multiple QDs of different wavelength to be used simultaneously(7, 8). We have employed two color QDs to investigate the drainage of the breast and arm to the axillary lymph nodes and found that some mice had independent drainage systems whereas other mice demonstrated nodes with"watershed" nodes, i.e. nodes that partly drained the breast and partly drained the limb(7, 8). Interestingly the node was divided into separate compartments according to its drainage. The implications of this anatomy for immunologic antigen processing are of potential interest. Additionally we have explored the use of up to 5 QDs of differing wavelength simultaneously in order to establish the complex lymphatic drainage patterns in the head and neck. These studies reveal that it is possible to identify drainage basins for particular regions and show areas of overlapping drainage. We have written a review on the subject of lymphatic imaging over the past year(1). Clinical Translation: We have been collaborating with Boston Children's Hospital to develop a dendrimer-based MR contrast agent for use in human children with primary lymphedema. As of September 07 the material for experimentation in a porcine model has been synthesized by Dr. Brechbiel's group and MIP personnel will assist Boston Children's Hospital in performing these studies. One important spin-off of this work is that we have generated a Gadolinium labeled albumin molecule. Each albumin is labeled with 10-15 Gadolinium atoms. As a consequence this is an excellent imaging agent on MRI and can be used as a surrogate marker. Drs. Ed Oldfield and Russ Lonser have expressed interest in using this agent as a surrogate marker for convection enhanced delivery (CED)agents in patients with brain tumors. Current CED therapy is limited by an inability to monitor the drug. Because Gd-Albumin demonstrates similar convection properties to the agent administered for CED (IL-13 PEG), it will be a useful surrogate marker. The JDC approved the GMP manufacture of Gd-Albumin for use in CED trials to be performed at the Clinical Center. The contract for the GMP production was awarded to the University of Kentucky-Lexington and the GMP synthesis is underway (Sept 07). 1. Barrett, T., Choyke, P. L., and Kobayashi, H. Imaging of the lymphatic system: new horizons. Contrast Media Mol Imaging, 1: 230-245, 2006. 2. Talanov, V. S., Regino, C. A., Kobayashi, H., Bernardo, M., Choyke, P. L., and Brechbiel, M. W. Dendrimer-based nanoprobe for dual modality magnetic resonance and fluorescence imaging. Nano Lett, 6: 1459-1463, 2006. 3. Kobayashi, H., Kawamoto, S., Bernardo, M., Brechbiel, M. W., Knopp, M. V., and Choyke, P. L. Delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging. J Control Release, 111: 343-351, 2006. 4. Koyama, Y., Talanov, V. S., Bernardo, M., Hama, Y., Regino, C. A., Brechbiel, M. W., Choyke, P. L., and Kobayashi, H. A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice. J Magn Reson Imaging, 25: 866-871, 2007. 5. Hama, Y., Bernardo, M., Regino, C. A., Koyama, Y., Brechbiel, M. W., Krishna, M. C., Choyke, P. L., and Kobayashi, H. MR lymphangiography using dendrimer-based contrast agents: a comparison at 1.5T and 3.0T. Magn Reson Med, 57: 431-436, 2007. 6. Xu, H., Regino, C. A., Bernardo, M., Koyama, Y., Kobayashi, H., Choyke, P. L., and Brechbiel, M. W. Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: new bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry. J Med Chem, 50: 3185-3193, 2007. 7. Kobayashi, H., Hama, Y., Koyama, Y., Barrett, T., Regino, C. A., Urano, Y., and Choyke, P. L. Simultaneous multicolor imaging of five different lymphatic basins using quantum dots. Nano Lett, 7: 1711-1716, 2007. 8. Hama, Y., Koyama, Y., Urano, Y., Choyke, P. L., and Kobayashi, H. Two-Color Lymphatic Mapping Using Ig-Conjugated Near Infrared Optical Probes. J Invest Dermatol, 2007.