Quantum Dots for NIR Fluorescence Imaging of Tumor Angiogenesis

描述（由申请人提供）：我们的长期目标是开发用于肿瘤血管生成和转移的多模态分子成像的适当探针。本申请的中间目的是开发用于体内α ν β 3整联蛋白表达的近红外（NIR）荧光成像的环状RGD肽缀合的量子点（QD）。细胞粘附分子av？3整合素在血管生成相关疾病中起关键作用，包括癌症、动脉粥样硬化、类风湿性关节炎、再狭窄和糖尿病视网膜病变。抑制AV？通过单克隆抗体、环状RGD肽拮抗剂和肽模拟物的3整联蛋白活性已显示诱导内皮细胞凋亡、抑制血管生成和增加内皮细胞单层通透性。我们和其他人还开发了适当标记的RGD肽和抗体，用于体内整联蛋白表达的MRI、超声、NIR荧光和放射性核素（PET和SPECT）成像。最近，我们第一次证明了RGD肽缀合的QD能够在异种移植模型中靶向整合素α v β 3的细胞外片段。然而，对这些纳米缀合物的进一步研究的热情被传统量子点的不利的体内动力学和基于镉的细胞毒性所减弱。我们建议在这里开发下一代的生物相容性量子点的近红外荧光成像。这些颗粒是超小的，具有薄的涂层材料，并且不是由镉硫属化物材料制成。这种类型的量子点适用于动物成像研究，并最终用于人类。首先，我们将开发一个小的非镉基量子点库，并将新的量子点与RGD肽缀合。新开发的非镉量子点的性能将与传统的镉基量子点进行比较。还将努力减少非特异性结合。其次，我们将评估QD-RGD在体内的整合素靶向功效。我们也将直接比较生物修饰的量子点与传统的分子成像探针标记的RGD肽与荧光染料。为了充分表征QD-RGD缀合物的生物分布和药代动力学，我们将用Cu（t1/2 = 12.7 h）和64 124 I（t1/2 = 4.2 d）放射性标记新开发的QD-RGD缀合物，用于组合NIR荧光和PET成像。最后，将对基于Cd和非Cd的QD-RGD缀合物进行急性和慢性毒性研究。我们希望新开发的非镉量子点具有很小或没有毒性，将适合临床翻译。这项研究对整合素av ² 3进行RGD肽修饰的成功可以扩展到QD多路复用，以提供有关细胞表面标志物（指示恶性肿瘤、肿瘤类型以及可能影响治疗程序）的实时信息。这些信息将是至关重要的荧光引导手术的敏感，特异性和实时术中可视化的分子特征的正常和病变的过程。荧光半导体纳米晶体（也称为量子点）在过去二十年中已经从纯电子材料科学发展到生物应用。已经应用了各种涂层技术以使QD具有生物相容性（水溶性和生物稳定性）。适当缀合的QD（通过抗体、蛋白质、肽或寡核苷酸）最常用于细胞生物学应用，包括DNA阵列技术、免疫荧光测定。然而，到目前为止，用于分子成像的QD的制备严重开发不足。此外，基于镉的细胞毒性进一步阻碍了用于体内应用的这种纳米构建物的开发。在此应用中，我们致力于开发用于肿瘤血管生成成像的非镉基超小量子点。我们将开发和表征QD-RGD缀合物库，然后进行体外和体内筛选。将对具有合适成像质量的QD进行急性和慢性毒性研究。该方法的成功将允许基于QD的探针用于荧光成像的临床转化。

DESCRIPTION (provided by applicant): Our long-term objective is to develop appropriate probes for multimodality molecular imaging of tumor angiogenesis and metastasis. Intermediate objective of this application is to develop cyclic RGD peptide conjugated quantum dots (QDs) for near-infrared (NIR) fluorescence imaging of av¿3 integrin expression in vivo. Cell adhesion molecule av¿3 integrin plays a key role in angiogenesis related diseases including cancer, atherosclerosis, rheumatoid arthritis, restenosis and diabetic retinopathy. Inhibition of av¿3 integrin activity by mAbs, cyclic RGD peptide antagonists, and peptidomimetics has been shown to induce endothelial apoptosis, to inhibit angiogenesis, and to increase endothelial monolayer permeability. Suitably labeled RGD peptides and antibodies have also been developed by us and others for MRI, ultrasound, NIR fluorescence, and radionuclide (PET and SPECT) imaging of integrin expression in vivo. Recently we demonstrated for the first time that RGD peptide conjugated QDs are able to target the extracellular segment of integrin av¿3 in an xenograft model. However, the enthusiasm for further studies of these nanoconjugates is mitigated by the unfavorable in vivo kinetics and cadmium-based cytotoxicity of the traditional quantum dots. We propose here to develop the next generation of biocompatible QDs for NIR fluorescence imaging. These particles are ultra small with thin coating material and are not made of cadmium chalcogenide materials. This type of QDs are suitable for animal imaging studies and eventually human use. First, we will develop a small library of non-Cd based QDs and conjugate the new QDs with RGD peptide. The performance of the newly developed non-Cd QDs will be compared with traditional Cd-based QDs. Efforts will also be spent to reduce non-specific binding. Second, we will evaluate the integrin targeting efficacy of QD-RGD in vivo. We will also directly compare biologically modified QDs with traditional molecular imaging probes by labeling RGD peptide with fluorescent dyes. In order to fully characterize the biodistribution and pharmacokinetics of the QD-RGD conjugates, we will radiolabel the newly developed QD-RGD conjugates with Cu (t1/2 = 12.7 h) and 64 124 I (t1/2 = 4.2 d) for combined NIR fluorescence and PET imaging. Finally, both Cd and non-Cd based QD-RGD conjugates will be subjected to acute and chronic toxicity studies. We expect that the newly developed non-Cd QDs with little or no toxicity will be amenable for clinical translation. The success of this study with RGD peptide modification for integrin av¿3 can be extended to QD multiplexing to provide the real-time information about cell surface markers (indicating malignancy, tumor type, and potentially influencing therapeutic procedure). Such information will be crucial for fluorescence-guided surgery by sensitive, specific, and real-time intraoperative visualization of molecular features of normal and diseased processes. Fluorescent semiconductor nanocrystals (a.k.a. quantum dots) have evolved over the last two decades from pure electronic materials science to biological applications. Various coating techniques have been applied to make QDs biocompatible (water-soluble and biologically stable). Suitably conjugated QDs (through antibody, proteins, peptides or oligonucleotides) are most commonly used in cell biology applications, including DNA array technology, immunofluorescence assays. However, preparation of QDs for molecular imaging has, so far, been severely under-developed. In addition, cadmium based cytotoxicity further hampered the development of such nanoconstructs for in vivo applications. In this application we thrive to develop non- cadmium based ultra small QDs for tumor angiogenesis imaging. We will develop and characterize a library of QD-RGD conjugates, followed by in vitro and in vivo screening. The QDs with suitable imaging quality will be subjected to acute and chronic toxicity studies. The success of this approach will allow clinical translation of QD based probe for fluorescence imaging.