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适合肿瘤血管生成和转移的多模态分子成像的探针。本应用程序的中间目标是开发环RGD肽共轭量子点（QDs）用于近红外（NIR）荧光成像av¿3整合素在体内的表达。细胞粘附分子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。单克隆抗体、环RGD肽拮抗剂和拟肽剂抑制av¿3整合素活性可诱导内皮细胞凋亡，抑制血管生成，增加内皮单层通透性。我们和其他人也开发了适当标记的RGD肽和抗体，用于MRI，超声，近红外荧光和放射性核素（PET和SPECT）成像体内整合素表达。最近，我们首次在异种移植模型中证明了RGD肽缀合的量子点能够靶向整合素3的细胞外片段。然而，传统量子点不利的体内动力学和镉基细胞毒性降低了对这些纳米偶联物进一步研究的热情。在此，我们建议开发用于近红外荧光成像的下一代生物相容性量子点。这些颗粒是超薄的涂层材料，并且不是由镉硫系材料制成的。这种类型的量子点适合于动物成像研究，并最终用于人类。首先，我们将建立一个小的非cd基量子点库，并将新的量子点与RGD肽偶联。新开发的非cd量子点的性能将与传统的基于cd的量子点进行比较。还将努力减少非特异性绑定。其次，我们将在体内评估QD-RGD的整合素靶向效果。我们还将通过荧光染料标记RGD肽，直接将生物修饰的量子点与传统的分子成像探针进行比较。为了充分表征QD-RGD偶联物的生物分布和药代动力学，我们将新开发的QD-RGD偶联物用Cu （t1/2 = 12.7 h）和64 124 I （t1/2 = 4.2 d）进行放射性标记，用于近红外荧光和PET联合成像。最后，Cd和非Cd基QD-RGD偶联物将进行急性和慢性毒性研究。我们期望新开发的毒性较小或没有毒性的非cd量子点可用于临床翻译。RGD肽修饰整合素av¿3的成功研究可以扩展到QD多路复用，以提供有关细胞表面标记物（指示恶性，肿瘤类型和潜在影响治疗程序）的实时信息。通过对正常和病变过程的分子特征进行敏感、特异和实时的术中可视化，这些信息将对荧光引导手术至关重要。荧光半导体纳米晶体（又名量子点）在过去的二十年中已经从纯电子材料科学发展到生物应用。为了使量子点具有生物相容性（水溶性和生物稳定性），应用了各种涂层技术。合适的偶联量子点（通过抗体、蛋白质、多肽或寡核苷酸）最常用于细胞生物学应用，包括DNA阵列技术、免疫荧光分析。然而，目前用于分子成像的量子点的制备还很不发达。此外，镉基细胞毒性进一步阻碍了这种纳米结构在体内应用的发展。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偶联物库，随后进行体外和体内筛选。具有合适成像质量的量子点将进行急性和慢性毒性研究。这种方法的成功将允许基于QD的荧光成像探针的临床翻译。

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.