Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada.
Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street W, Hamilton, ON L8S 4L8, Canada.
ACS Appl Mater Interfaces. 2021 Mar 3;13(8):9412-9424. doi: 10.1021/acsami.0c16666. Epub 2021 Jan 4.
: An important clinical question in the determination of the extent of thrombosis-related vascular conditions is the identification of blood clot location. Fibrin is a major molecular constituent of blood clots and can, therefore, be utilized in molecular imaging. In this proof-of-concept study, we sought to prepare a fibrin-targeting magnetic resonance imaging contrast agent, using a Gd(III)-loaded fibrinogen aptamer (FA) chelate conjugate (Gd(III)-NOTA-FA) (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid), to endow the ability to specifically accumulate at the location of blood clots, thereby enhancing contrast capabilities. : The binding affinity of FA for fibrin was confirmed by fluorescence microscopy and microscale thermophoresis. The preparation and effective loading of the chelate-aptamer conjugates were confirmed by mass spectrometry and a xylenol orange colorimetric test. Longitudinal and transverse relaxivities and the effects of target binding were assessed using T1- and T2-map sequences at 7 T. T1- and T2-weighted images were acquired after blood clots were treated with Gd(III)-NOTA-FA. Collagen was used as the protein control, while an unrelated aptamer sequence, FB139, was used as the aptamer control. : FA demonstrated a high affinity and selectivity toward the polymeric protein, with a of 16.6 nM, confirming an avidity over fibrinogen. The longitudinal (r1) and transverse (r2) relaxivities of Gd(III)-NOTA-FA demonstrated that conjugation to the long aptamer strand shortened T1 relaxation times and increased T2 relaxation times (3.04 and 38.7 mM s, respectively). These effects were amplified by binding to the fibrin target (1.73 and 46.5 mM s, respectively). In vitro studies with thrombin-polymerized human blood (clots) in whole blood showed an unexpected enhancement of signal intensity (hyperintense) produced exclusively at the location of the clot during the T2-weighted scan, while the presence of fibrinogen within a whole blood pool resulted in T1 signal intensity enhancement throughout the pool. This is advantageous, as simply reversing the type of a scan from a typical T1-weighted to a T2-weighted would allow to selectively highlight the location of blood clots. : Gd(III)-NOTA-FA can be used for molecular imaging of thrombi, through fibrin-targeted delivery of contrast to the location of blood clots in T2-weighted scans.
在确定与血栓相关的血管状况的范围时，一个重要的临床问题是识别血栓的位置。纤维蛋白是血栓的主要分子成分，因此可以用于分子成像。在这项概念验证研究中，我们试图制备一种纤维蛋白靶向磁共振成像对比剂，使用负载钆（III）的纤维蛋白原适体（FA）螯合物（Gd（III）-NOTA-FA）（NOTA=1,4,7-三氮杂环壬烷-1,4,7-三乙酸），使其能够特异性地聚集在血栓的位置，从而增强对比能力。
FA 对纤维蛋白的结合亲和力通过荧光显微镜和微尺度热泳证实。通过质谱和二甲酚橙比色试验证实了螯合物-适体缀合物的制备和有效负载。在 7T 下使用 T1-和 T2-图序列评估纵向和横向弛豫率以及靶结合的影响。在处理 Gd（III）-NOTA-FA 后，获得血栓的 T1-和 T2-加权图像。胶原蛋白用作蛋白质对照，而不相关的适体序列 FB139 用作适体对照。
FA 对聚合蛋白表现出高亲和力和选择性，解离常数（Kd）为 16.6 nM，证实了对纤维蛋白原的亲和力。Gd（III）-NOTA-FA 的纵向（r1）和横向（r2）弛豫率表明，与长适体链的缀合缩短了 T1 弛豫时间并增加了 T2 弛豫时间（分别为 3.04 和 38.7 mM s）。通过与纤维蛋白靶标结合，这些效应得到放大（分别为 1.73 和 46.5 mM s）。在全血中的凝血酶聚合的人血（血栓）的体外研究中，在 T2 加权扫描期间，仅在血栓位置产生信号强度（高信号）的意外增强，而全血池中的纤维蛋白原导致整个池的 T1 信号强度增强。这是有利的，因为只需将扫描类型从典型的 T1 加权扫描反转到 T2 加权扫描，就可以选择性地突出血栓的位置。
Gd（III）-NOTA-FA 可用于血栓的分子成像，通过将对比剂靶向递送至 T2 加权扫描中的血栓位置来实现。
