Kumar Anand T N, Rice William L, López Jessica C, Gupta Suresh, Goergen Craig J, Bogdanov Alexei A
A. Martinos' Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown MA 02129.
Department of Radiology and the Laboratory of Molecular Imaging Probes, University of Massachusetts Medical School, Worcester, MA 01655.
ACS Sens. 2016 Apr 22;1(4):427-436. doi: 10.1021/acssensors.5b00252. Epub 2016 Feb 9.
Enzymatic activity sensing in fluorescence lifetime (FLT) mode with "self-quenched" macromolecular near-infrared (NIR) sensors is a highly promising strategy for imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ=0.1-0.2 min) FLT increase (Δτ=0.25 ns) upon model proteinase-mediated hydrolysis , lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence de-quenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were de-quenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8-10kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of "built-in" dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.
利用“自猝灭”大分子近红外（NIR）传感器以荧光寿命（FLT）模式进行酶活性传感是一种极有前景的蛋白水解成像策略。然而，此类基于底物的近红外传感器中FLT变化的机制尚未得到研究。我们通过将近红外荧光团IRDye 800CW连接到具有不同甲氧基聚乙二醇化程度的甲氧基聚乙二醇和聚赖氨酸（MPEG-gPLL）大分子接枝共聚物上，合成了两种类型的传感器，并研究了它们在胰蛋白酶、弹性蛋白酶、纤溶酶和组织蛋白酶（B、S、L、K）作用下的片段化情况。我们确定了此类近红外传感器在FLT模式下的效率取决于传感器组成。虽然高度甲氧基聚乙二醇化的MPEG-gPLL在模型蛋白酶介导的水解作用下显示出快速（τ = 0.1 - 0.2分钟）的FLT增加（Δτ = 0.25纳秒），但较低的甲氧基聚乙二醇化密度则不会导致这种FLT增加。近红外传感器荧光去猝灭的温度依赖性表明MPEG-gPLL连接的荧光团存在混合动态/静态猝灭模式。我们进一步证明，尽管由于静态猝灭的消除，大部分与传感器连接的荧光团被去猝灭，但蛋白水解介导的高度甲氧基聚乙二醇化近红外传感器中一部分短（8 - 10kD）带负电荷片段的缺失是导致猝灭近红外传感器中FLT快速增加现象的最可能事件。因此，优化大分子近红外传感器的“内置”动态猝灭元件是提高其在FLT模式下响应的潜在途径。
