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

描述（由申请人提供）：炎症反应的不良激发是许多疾病（如糖尿病、动脉粥样硬化和哮喘）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鉴定炎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从疾病动物模型切除的组织的分析上。在该提案中，我们描述了一个平台的开发，该平台用于使用量子点纳米晶体和视网膜荧光显微镜在患病动物模型中实时、体内分析炎症的多种细胞和分子介质。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。这些特性包括相对于常规染料更高的量子效率、抗褪色性、可由一个波长激发的窄且尺寸可调的发射光谱，以及对蛋白质和聚合物的表面工程的适应性，以获得最佳稳定性和靶向。第二个使能技术是我们在体内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可达性。为了验证我们提出的技术的潜力，在目标1中，我们寻求优化成像模式/纳米探针设计，以同时检测炎症ICAM-1、VCAM-1、PECAM-1和E-选择素的治疗上显著的生物标志物，并观察它们在糖尿病大鼠模型中视网膜血管中的相对分子表达水平。此外，我们试图调整目标1中建立的探针/荧光成像设计，以在同一动物模型中体内真实的时间跟踪多个白细胞亚群。我们的初步工作表明，这种技术的特异性标记和在体内检测的内皮细胞表面标志物ICAM-1，VCAM-1和PECAM-1在体内，以及移动，在体内标记的中性粒细胞在视网膜循环，具有高的空间和时间分辨率和高信号背景比的潜力。我们的初步研究的一个亮点是糖尿病中VCAM-1和ICAM-1上调的首次体内验证。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，而且在其他疾病如癌症和眼部疾病中也是如此。

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.