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.

项目摘要/摘要 酶在生物学过程中起关键作用，并测量细胞和组织中的酶活性 是我们对生理学和病理生理学的理解的基础。在体内测量酶活性 在完整的活组织中提供背景 酶活性的时间差异。但是，关于酶活性的大多数研究都是 由于我们无法观察到具有高灵敏度和高度的酶活性，因此可以在体外和体内方法 深层组织中的分辨率。基于荧光的技术提供了非侵入性高分辨率的观点，可查看分子 ULAR或形态学信息，但通常受到组织渗透有限的阻碍。在光学tis-中进行成像 在短波红外（SWIR; 900-1700 nm）中的起诉窗户可实现更深的组织渗透并改善 由于生物材料的吸收和散射减少，分辨率比可见光或NIR光。甚至在 理想的波长，光致发光成像需要入射光以激发荧光团，从而导致 轻衰减都进出组织。基于荧光素酶的生物发光成像消除了 需要外部激发，从而降低距离光必须穿过组织以及消除 Nating自动荧光。尽管对于许多体内成像应用而优选生物发光，但有 没有关于SWIR发射荧光素酶的报道，将这种方法限制为较少首选的可见波长或NIR 发射<750 nm的探针。在此提案中，生物发光能量转移（BRET）从荧光素酶到Swir- 排放半导体量子点（QD）会产生自我缩放的Swir发射造影剂。我们支持 构成第一种酶 - 发射，自我添加的量子点的开发 （QD）。多路复用参考和可裂解体内比值计的酶探针将校准 酶活性探针的局部浓度，并由我们的高光谱Swir临床前促进 Ager。我们将通过可视化MMP-16的活性来测试我们的传感器，MMP-16的活性是肝病的细胞表面生物标志物 与纤维化，肝炎和肝细胞癌（HCC）相关的进展。测试我们的假设 SIS与自我刷新，发射对比剂的双重探针成像将使空间和温度能够 孔酶活性图，我们将在体外设计，表征和优化BRET探针。并行，我们 将使用组织幻像比较使用BRET与Pho-的信噪比（S/N）和图像分辨率进行比较 耐受发光激发机制。最后，我们将证明传感器在phan-中的功能 toms和肝肝硬化的小鼠模型。成功完成该项目将导致传感器CA- 报告MMP-16活性在肝脏深处，具有高分辨率和高灵敏度。证明这一点 采取无创酶活性的非侵入性，高分辨率图像的新方法将使广泛的AR- 科学界在病理生理学中的作用的新研究。