Intracellular In vivo Imaging

迄今为止，体内分子显像剂专门针对细胞表面或微环境。然而，癌细胞分化为癌细胞与正常细胞的高度特异性变化大多发生在细胞内。因此，挑战在于开发能够报告胞浆内变化但仍能在体内成像的药物。实现这一目标的第一步是将显像剂靶向到细胞表面，这需要对细胞表面标记物具有亲和力。然后配体必须通过内吞作用内化，然后结合到适当的位点，在那里它被“激活”。这些要求对合成化学提出了很高的要求，因为分子结构必须具有多种功能。我们正在开发“智能”可激活的光学结构，只有当它们内化到细胞质中时才会发出荧光。利用一系列商业上可用的染料，这些染料与靶向化合物结合，然后在特定的细胞内条件下（如较低的pH值和存在特定的酶活性）进行修饰，发出荧光，我们正在朝着细胞内体内成像的目标取得进展。这项工作是与东京大学化学系的Urano教授合作进行的。在过去的一年里，我们在这一领域取得了相当大的进展，证明了基于BODIPY和罗丹明骨架制造高度可激活的光学显像剂是可能的。我们也在努力开创多模式显像剂；可以在光学相机、PET、MR或放射性核素相机上看到的试剂。正在设计的药物具有高度的生物相容性，其中一些成分已经在人体中使用。例如，我们用罗丹明绿（GSA- rhg）标记的半乳糖血清白蛋白（GSA）在癌细胞内迅速内化，可能作为人类使用的药物是可行的。我们正在开发活化荧光分子显像剂，并在过去一年中取得了一些成功。然而，我们将继续寻求一种只在癌细胞内激活而不在其他正常细胞内激活的解决方案。除了GSA作为靶向配体外，我们还使用市售抗体，如曲妥珠单抗和西妥昔单抗，这些抗体将实现靶向、结合和内化。具体来说，我们已经将曲妥珠单抗与自猝灭吲哚菁绿（ICG）联合使用，ICG是一种FDA批准的近红外（NIR）染料，用于靶向原位肿瘤。当抗体与其同源受体结合时，它被内化，释放出ICG，然后开始发出荧光。这个结构的两个组成部分都是fda批准的，所以理论上这项技术可以很容易地转化为临床。同时对多个目标成像的能力促使我们探索多激发和多发射相机。我们曾希望单一激发光能够激活不同波长的多个荧光团，但事实证明这是不现实的。取而代之的是，我们使用多波长的激发光，使用新的Maestro相机。这使我们能够同时在近红外成像多达4个目标，在临床应用中非常有前景。此外，我们正在开发带有荧光受体的光纤示波器，使用小型光纤示波器可以对非常小的区域进行经皮检查。最近，我们已经证明，通过使用高度调谐的实时摄像机，可以对未麻醉的活体小鼠进行成像。在不久的将来，开发多靶点多彩色成像来更好地表征肿瘤是可能的。最近，我们已经使用Halotag技术来进行体内成像。目前，如果研究人员想要用荧光标记物（如绿色荧光蛋白（GFP））标记细胞，他们必须用合适的基因转染细胞。如果他们对彩色成像的需求发生变化（即，他们需要将细胞标记为红色，以避免与发出绿色荧光的药物竞争），他们必须重新获得表达红色荧光蛋白（RFP）的细胞系。Halotag是一种基因结构，可以在细胞表面产生一种特殊的、非天然的酶表达。然后，Halo配体可以贪婪地结合这种酶。通过将不同颜色的荧光团附着到每个Halo配体上，可以改变细胞的颜色，而无需重新衍生细胞系，这是多色实验的潜在优势。此外，我们实验室最近的结果表明，通过简单地将靶向Halo配体与另一个荧光团交换，可以获得出色的成像结果。这种方法为类似的实验增加了灵活性。

To date, in vivo molecular imaging agents specifically target the cell surface or microenvironment. However, most of the highly specific changes of cancer cells that differentiate cancers cells from normal cells occur intracellularly. The challenge, therefore, is to develop agents that report intracytoplasmic changes yet still are capable of being imaged in vivo. The first step in achieving this goal is to target the imaging agent to the cell surface which requires affinity for a cell surface marker. The ligand must then be internalized by endocytosis and then bind to the appropriate site whereupon it "activates". These requirements place large demands on synthetic chemistry since the molecular construct must have multiple functionalities. We are developing "smart" activatable optical constructs which only fluoresce when they are internalized to the cytoplasm. Using a series of commercially available dyes that are bound to targeting compounds and then modified to fluoresce under specific intracellular conditions such as lower pH and in the presence of specific enzymatic activity we are making progress toward the goal of intracellular in vivo imaging. This work is being performed in collaboration with Prof. Urano from the University of Tokyo Chemistry Department. Over the past year we have made considerable advances in this area by proving that it is possible to create highly activatable optical imaging agents based on the BODIPY and Rhodamine backbones. We are also pioneering efforts to create multimodal imaging agents; agents that can be seen on both optical cameras as well as PET, MR or radionuclide cameras. The agents being designed are highly biocompatible and elements have already been used in humans. For intance, the agent Galactosylserum Albumin (GSA) which we have labeled with Rhodamine Green (GSA-RhG) is internalized rapidly within cancer cells and may be viable as an agent for human use. We are developing activated fluorescent molecular imaging agents and have a number of successes over the year. However, we continue to pursue a solution that will activate only within cancer cells and not within other, normal cells. In addition to GSA as a targeting ligand we are employing commercially available antibodies such as trastuzumab and cetuximab that will enable targeting, binding and internalization. Specifically, we have used trastuzumab in combination with a self quenched indocyanine green, ICG, an FDA approved Near InfraRed (NIR) dye to target in situ tumors. When the antibody binds its cognate receptor, it is internalized releasing the ICG which then begins to fluoresce. Both components of this construct are FDA-approved so in theory this technique could be translated clinically fairly easily. The ability to image multiple targets simulanteously led us to explore multiexcitation and multiemission cameras. We had hoped that a single excitation light would be able to activate multiple fluorophores at differing wavelengths but this proved to be unrealistic. Instead, we use multiple wavelength excitation light using a new Maestro camera. This has allowed us to simultaneously image up to 4 targets in the near infrared and is very promising for clinical application. Additionally, we are developing fiberoptic scopes with fluorescence receptors to allow very small areas to be examined percutaneously using small fiber-based scopes. Recently, we have demonstrated that is possible to image live unanesthetized mice by using a highly tuned real time camera. It may be possible in the near future to develop multi-targeted multi color imaging to better characterize tumors. Recently, we have used the Halotag technology to perform in vivo imaging. Currently, if researchers want to label cells with a fluorescent marker (such as Green Fluorescent Protein (GFP)), they must transfect the cell with a suitable gene. If their needs for color imaging change (i.e., they need to label the cells red so as not to compete with a drug that fluoresces in the green) they must rederive a cell line that expresses Red Fluorescent Protein (RFP). Halotag is a genetic construct that leads to a special, non-natural enzyme expression on the cell surface. A Halo ligand can then bind this enzyme avidly. By attaching different color fluorophores to each Halo ligand it is possible to change the colors of the cells without rederiving the cell line, a potential advantage in multicolor experiments. Moreover, recent results in our lab demonstrate that is possible to get excellent imaging results by simply exchanging the targeting Halo ligand with another fluorophore. This method should add flexibility to similar experiments.