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

项目摘要/摘要 酶在生物过程中起着关键作用，并测量细胞和组织中的酶活性 是我们理解生理学和病理生理学的基础。体内酶活性的测定 在完整的活组织中提供背景，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是有限的- 由于我们不能高灵敏度和高效率地观察酶活性，我们采用了体外和体外的方法 在深层组织中的分辨率。基于荧光的技术提供了对分子的非侵入性、高分辨率的观察- 形态或形态信息，但往往因有限的组织穿透而受阻。光学组织中的成像 短波红外(SWIR；900-1700 nm)中的SUE窗口可实现更深层次的组织穿透并改善 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。即使是在 理想波长的光致发光成像需要入射光来激发荧光团，从而导致 进入和离开组织的光衰减。基于荧光素酶的生物发光成像消除了 需要外部激发，从而减少了光必须穿过组织的距离，并消除了 产生自体荧光。虽然生物发光是许多活体成像应用的首选，但也有 没有关于SWIR发光荧光素酶的报道，将这种方法限制在较不受欢迎的可见光波长或NIR 发射&lt；750 nm的探头。在这个方案中，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)产生自发光的SWIR发射造影剂。我们支持- 提出了第一个酶传感、SWIR发射、自发光量子点的开发 (量子点数)。用于比率测量的体内成像的多路参考和酶可切割探针将校准 酶活性与探针的局部浓度有关，并由我们的高光谱SWIR临床前临床应用促进。 阿格。我们将通过可视化肝脏疾病的细胞表面生物标记物--基质金属蛋白酶-16的活性来测试我们的传感器 进展与纤维化、肝炎和肝细胞癌(HCC)相关。为了测试我们的假说- SIS认为，带有自发光、SWIR发射造影剂的双探针成像将使空间和温度成像成为可能。 我们将在体外设计、表征和优化BRET探针。同时，我们 将使用组织模体来比较信噪比(S/N)和图像分辨率(使用BRET和PHO- 热释光激发机制。最后，我们将在Phan中演示我们的传感器的功能- 汤姆斯和一只小鼠的肝硬变模型。该项目的成功完成将导致一个传感器案例- 能以高分辨率和高灵敏度报告肝脏深处的基质金属蛋白酶-16活性。证明了这一点 非侵入性、高分辨率拍摄深层组织酶活性图像的新方法将使广泛的AR。 科学界对酶在病理生理学中的作用的新研究。

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.