Multi-spectral quantum dot-based retinal imaging of molecular expression in vivo

描述(由申请人提供)：炎症反应的不良刺激是糖尿病、动脉粥样硬化和哮喘等多种疾病的有害特征，因此影响了大量人群。目前用于在细胞和分子水平上确定炎症性疾病的关键标志物的策略通常集中在体外细胞培养研究或对从疾病动物模型中切除的组织进行分析。在这项建议中，我们描述了一个平台的开发，用于使用量子点纳米晶体和视网膜荧光显微镜对疾病动物模型中的多种细胞和分子炎症介质进行实时、活体分析。我们的设计与以前研究体内炎症的方法不同，因为它是通过利用赋予量子点的独特和高度理想的光学特性来实现的。这些特性包括比传统染料更高的量子效率，抗衰落，发射光谱窄且大小可调，均可由一个波长激发，以及可用于蛋白质和聚合物的表面工程，以实现最佳稳定性和靶向性。第二项使能技术是我们在活体视网膜荧光成像方面的实验室专业知识，它为体内血管系统提供了独特的连续光学访问。为了验证我们建议的技术的潜力，在目标1中，我们试图优化成像模式/纳米探针设计，以同时检测具有治疗意义的炎症生物标记物ICAM-1、VCAM-1、PECAM-1和E-选择素，并观察它们在糖尿病大鼠模型视网膜血管中的相对分子表达水平。此外，我们试图采用目标1中建立的探针/荧光成像设计，在同一动物模型中实时跟踪体内的多个白细胞亚群。我们的初步工作表明，该技术具有高时空分辨率和高信本比的潜在潜力，用于体内特异性标记和在体检测内皮表面标记ICAM-1、VCAM-1和PECAM-1，以及视网膜循环中运动的、在体标记的中性粒细胞。我们初步研究的一个亮点是首次在体内验证VCAM-1和ICAM-1在糖尿病中的上调。这项技术有可能阐明复杂的、细胞和分子事件，因为它们不仅在炎症中实时发生，而且在癌症和眼疾等其他疾病中也发生。

DESCRIPTION (provided by applicant): Undesirable provocation of the inflammatory response is a detrimental feature of numerous diseases such as diabetes, atherosclerosis, and asthma and thus impacts a significant population. Current strategies utilized to identify key markers of inflammatory disease at the cellular and molecular level have often been centered on in vitro cell culture studies or analyses of tissue excised from animal models of disease. In this proposal we describe the development of a platform for the real-time, in vivo analysis of multiple cellular and molecular mediators of inflammation in diseased animal models using quantum dot nanocrystals and retinal fluorescence microscopy. Our design differs from previous approaches to study inflammation in vivo in that it is enabled by the harnessing of unique and highly-desirable optical properties conferred to quantum dots. These properties include higher quantum efficiency relative to conventional dyes, a resistance to fading, narrow and size-tunable emission spectra all excitable by one wavelength, and amenability to surface engineering of proteins and polymers for optimum stability and targeting. The second enabling technology is our laboratory expertise in in vivo retinal fluorescence imaging which provides unique continuous optical accessibility to the in vivo vasculature. To validate the potential of our proposed technique, in Aim 1 we seek to optimize an imaging modality / nanoprobe design to simultaneously detect the therapeutically-significant biomarkers of inflammation ICAM-1, VCAM-1, PECAM-1, and E-selectin, and to observe their relative molecular expression levels in the retinal vasculature in a rat model of diabetes. Furthermore, we seek to adapt the probe/fluorescence imaging design established in Aim 1 to track multiple leukocyte subsets in real time in vivo in the same animal model. Our preliminary work demonstrates the potential of this technique for the specific labeling and in vivo detection of the endothelial surface markers ICAM-1, VCAM-1 and PECAM-1 in vivo, as well as moving, in vivo-labeled neutrophils in the retinal circulation, with high spatial and temporal resolution and high signal to background ratios. A highlight of our preliminary studies was the first in vivo validation of VCAM-1 and ICAM-1 upregulation in diabetes. This technology has the potential to elucidate complex, cellular and molecular events as they occur in real-time not only in inflammation, but in other diseases such as cancer and ocular disorders as well.