In Vivo Mapping of Enzyme Activity using SWIR-emitting, Self-illuminating Quantum Dot Sensors

使用短波红外发射、自发光量子点传感器绘制酶活性体内图谱

• 批准号: 10762565
• 负责人: Allison Marie Dennis
• 金额: $ 23.89万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762565
• 关键词: Vivo; Mapping; Enzyme; Activity; using

Project Summary/Abstract Enzymes play a critical role in biological processes, and measurements of enzyme activity in cells and tissues are foundational to our understanding of physiology and pathophysiology. Measuring enzyme activity in vivo provides context within the intact living tissue, enabling organ- or organism-level understanding of the spatial and temporal differences in enzyme activity. However, most research regarding enzyme activity has been lim- ited to in vitro and ex vivo methods due to our inability to observe enzyme activity with high sensitivity and high resolution in deep tissues. Fluorescence-based techniques offer a non-invasive, high resolution look at molec- ular or morphological information but are often hampered by limited tissue penetration. Imaging in optical tis- sue windows in the short-wave infrared (SWIR; 900-1700 nm) enables deeper tissue penetration and improved resolution than visible or NIR light due to reduced absorption and scattering by biological materials. Even at ideal wavelengths, photoluminescence imaging requires incident light to excite the fluorophore, resulting in light attenuation both going in and out of the tissue. Luciferase-based bioluminescence imaging eliminates the need for external excitation, thereby reducing the distance light must traverse through tissue as well as elimi- nating autofluorescence. While bioluminescence is preferred for many in vivo imaging applications, there are no reports of SWIR-emitting luciferases, limiting this approach to less preferred visible wavelengths or NIR probes emitting < 750 nm. In this proposal, bioluminescence energy transfer (BRET) from luciferases to SWIR- emitting semiconductor quantum dots (QDs) generate self-illuminating SWIR-emitting contrast agents. We pro- pose the development of the first enzyme-sensing, SWIR-emitting, self-illuminating quantum dots (QDs). Multiplexing reference and enzyme-cleavable probes for ratiometric in vivo imaging will calibrate the enzyme activity to the local concentration of probes and is facilitated by our hyperspectral SWIR preclinical im- ager. We will test our sensor by visualizing the activity of MMP-16, a cell-surface biomarker of liver disease progression that is correlated with fibrosis, hepatitis, and hepatocellular carcinoma (HCC). To test our hypothe- sis that dual probe imaging with self-illuminating, SWIR-emitting contrast agents will enable spatial and tem- poral enzyme activity maps, we will design, characterize, and optimize the BRET probes in vitro. In parallel, we will use tissue phantoms to compare the signal-to-noise ratios (S/N) and image resolution using BRET vs. pho- toluminescence excitation mechanisms. Finally, we will demonstrate the functionality of our sensor in phan- toms and in a mouse model of liver cirrhosis. Successful completion of this project would result in a sensor ca- pable of reporting MMP-16 activity deep in the liver with high resolution and high sensitivity. Demonstrating this new way to take non-invasive, high resolution images of enzyme activity in deep tissues will enable a wide ar- ray of new studies into the role of enzymes in pathophysiology by the scientific community.

项目概要/摘要 酶在生物过程以及细胞和组织中酶活性的测量中发挥着关键作用 是我们理解生理学和病理生理学的基础。测量体内酶活性 提供完整活组织内的背景，使器官或有机体水平的空间理解成为可能 以及酶活性的时间差异。然而，大多数关于酶活性的研究都受到限制。 由于我们无法以高灵敏度和高灵敏度观察酶活性，因此只能采用体外和离体方法 深部组织的分辨率。基于荧光的技术提供了非侵入性、高分辨率的分子分析 形状或形态信息，但经常受到有限的组织渗透的阻碍。光学成像中 短波红外（SWIR；900-1700 nm）的苏窗口可实现更深的组织穿透并改善 由于生物材料的吸收和散射减少，分辨率高于可见光或近红外光。即使在 在理想波长下，光致发光成像需要入射光来激发荧光团，从而产生 进出组织的光衰减。基于荧光素酶的生物发光成像消除了 需要外部激发，从而减少光必须穿过组织的距离，并消除 自发荧光。虽然生物发光是许多体内成像应用的首选，但也有 没有关于发射短波红外荧光素酶的报道，限制了这种方法用于不太优选的可见波长或近红外 发射< 750 nm 的探针。在该提案中，从荧光素酶到 SWIR 的生物发光能量转移（BRET） 发射半导体量子点（QD）产生自发光短波红外发射造影剂。我们亲 开发出第一个酶感应、短波红外发射、自发光量子点 （QD）。多重参考探针和酶切探针用于比例体内成像将校准 我们的高光谱 SWIR 临床前成像促进了酶活性对探针局部浓度的影响 老化。我们将通过观察 MMP-16（肝病细胞表面生物标志物）的活性来测试我们的传感器 与纤维化、肝炎和肝细胞癌 (HCC) 相关的进展。为了检验我们的假设—— 事实是，使用自发光、短波红外发射造影剂的双探头成像将能够实现空间和温度 口腔酶活性图，我们将在体外设计、表征和优化 BRET 探针。与此同时，我们 将使用组织模型来比较信噪比 (S/N) 和使用 BRET 与 pho 的图像分辨率 发光激发机制。最后，我们将演示我们的传感器的功能 汤姆和肝硬化小鼠模型。该项目的成功完成将导致传感器ca- 高分辨率和高灵敏度报告肝脏深处 MMP-16 活性的表格。证明这一点 拍摄深层组织中酶活性的非侵入性高分辨率图像的新方法将使广泛的研究成为可能。 科学界对酶在病理生理学中的作用进行了一系列新研究。