QD-BRET nanosensors for protease detection and imaging

描述(申请人提供)：原发肿瘤转移到远处器官部位是导致高癌症死亡率的主要原因。这种迁移过程被认为是由包括基质金属蛋白酶(MMPs)在内的一系列水解酶帮助的。MMPs通过选择性地降解细胞外基质和非基质蛋白来定义细胞环境，在大多数正常组织中很低或检测不到，但在大多数恶性肿瘤中显著增加。它们的表达程度与肿瘤分期、侵袭、转移和血管生成有关。然而，最近对基质金属蛋白酶抑制剂的临床试验结果令人失望，强调有必要更好地了解这一多功能蛋白水解酶家族在从起始、血管生成到远处器官部位转移灶的建立和生长的多个步骤中促进肿瘤生长和进展的机制。本研究建议开发一种基于纳米技术的新型传感和成像系统，用于对生物样本和活体中的MMPs进行灵敏、准确和多重的检测和成像，并应用这一新技术来研究MMPs在肿瘤形成、生长和进展中的功能作用。这一新平台是基于量子点和生物发光共振能量转移(BRET)的。量子点是微小的荧光半导体纳米晶体，可以用定义的发射波长产生光谱，并用于多重检测。在量子点-Bret过程中，量子点可以通过发光蛋白质的共振能量传递过程发光。本研究将开发一系列基于QD-Bret平台的纳米传感器来检测和成像三种与肿瘤转移密切相关的重要蛋白酶：明胶酶A，负责降解细胞外基质的主要成分IV型胶原；基质溶素，最小的成员，具有广泛的蛋白分解活性；以及尿激酶型纤溶酶原激活物(UPA)，涉及MMPs的激活。本研究有三个具体目的：1)建立生物样品(细胞培养液和小鼠血清)中MMP2、MMP7和uPA的QD-RBET检测平台；2)在小鼠移植瘤模型中复合检测MMP2、MMP7和UPA的活性；3)通过小干扰RNA检测UPA、MMP2和MMP7的抑制效果，并探讨三者之间的交叉激活关系。这种QD-Bret传感和成像纳米平台对于生物分子的早期检测以及对肿瘤活动和功能的在体实时监测都将是无价的。这里制定的战略也可以推广到许多其他目标。深入了解活体肿瘤组织和正常组织蛋白分解活性的差异，将有助于我们对肿瘤转移的理解，并有助于抗转移治疗的发展。 与公共健康相关：拟议的研究旨在开发新的纳米技术来检测和成像关键酶，以促进肿瘤从原发部位向远程器官的转移。这项新的纳米技术将允许高度敏感地检测生物样本中的这些酶分子，以帮助及早发现癌症。深入了解活体肿瘤组织与正常组织酶活性的差异，将有助于我们对肿瘤转移的认识，并有助于抗转移治疗的发展。

DESCRIPTION (provided by applicant): Metastasis of primary tumors to distant organ sites is mainly responsible for high cancer fatality. This migration progress is believed to be assisted by a family of hydrolytic enzymes including matrix metalloproteinases (MMPs). MMPs define the cellular environment through selectively degrading both extracelluar matrix and non-matrix proteins, and are low or undetectable in most normal tissues, but substantially increased in the majority of malignant tumors. The extent of their expression has been shown to be related to tumor stage, invasiveness, metastasis and angiogenesis. The recent disappointing results of clinical trails of MMP inhibitors, however, emphasize the need for better understanding of the mechanism by which this family of multifunctional proteases contributes to multiple steps of tumor growth and progression, from initiation, angiogenesis, and the establishment and growth of metastatic lesion in distant organ sites. This research proposes to develop a novel nanotechnology-based sensing and imaging system for sensitive, accurate, and multiplex detection and imaging of MMPs in biological samples and in living subjects and apply this new technique to investigate the functional roles of MMPs in the tumor formation, growth and progression. This new platform is based on quantum dots (QDs) and bioluminescence resonance energy transfer (BRET). Quantum dots are tiny fluorescent semiconductor nanocrystals that can be produced with a spectrum of defined emission wavelengths and used for multiplex detection. QDs can emit light via the resonance energy transfer process from a light-emitting protein in the process of QD-BRET. This research will develop a series of nanosensors based this QD-BRET platform to detect and image three important proteases that highly involve in tumor metastasis: MMP-2, gelatinase A responsible for the degradation of type IV collagen, the main component of extracellular matrix; MMP-7, matrilysin, the smallest member with broad proteolytic activity; and urokinase-type plasminogen activator (uPA) that involves the activation of MMPs. There are three specific aims: 1) to establish the QD-RBET detection platform for multiplexing analysis of MMP-2, MMP-7, and uPA in biological samples (in cell medium and mouse serum); 2) to multiplex image MMP-2, MMP-7, and uPA activity in xenografted tumors in a mouse model; 3) to image the inhibition efficacy of uPA, MMP-2 and MMP-7 by small interfering RNA and to probe the cross-activation relationship among the three proteases. This QD- BRET sensing and imaging nanoplatform will be invaluable for both early detection of biomakers and for in vivo real-time monitoring of tumor activity and function. The strategy developed here can also be extended to many other targets as well. A sophisticated understanding of the differences in proteolytic activity between tumor and normal tissues in living subjects will advance our understanding of cancer metastasis and help develop anti- metastasis therapy. PUBLIC HEALTH RELEVANCE: The proposed research aims to develop novel nanotechnology to detect and image critical enzymes that facilitate tumor migration from primary sites to remote organs. This new nanotechnology will allow highly sensitive detection of these enzyme molecules in biological samples to help early detection of cancers. A sophisticated understanding of the differences of enzyme activity between tumor and normal tissues in living subjects will advance our understanding of cancer metastasis and help develop antimetastasis therapy.