QD-BRET nanosensors for protease detection and imaging

描述（由申请人提供）：原发性肿瘤转移到远处器官部位是癌症高死亡率的主要原因。这种迁移过程被认为是由一个家庭的水解酶，包括基质金属蛋白酶（MMP）的协助。MMPs通过选择性降解细胞外基质和非基质蛋白来限定细胞环境，并且在大多数正常组织中是低的或不可检测的，但在大多数恶性肿瘤中显著增加。它们的表达程度与肿瘤的分期、侵袭性、转移和血管生成有关。然而，最近MMP抑制剂的临床试验结果令人失望，强调需要更好地理解该多功能蛋白酶家族促进肿瘤生长和进展的多个步骤的机制，从起始，血管生成，以及远处器官部位转移性病变的建立和生长。本研究提出开发一种新型的基于纳米技术的传感和成像系统，用于生物样品和活体中MMPs的灵敏，准确和多重检测和成像，并应用这种新技术来研究MMPs在肿瘤形成，生长和进展中的功能作用。这个新的平台是基于量子点（QD）和生物发光共振能量转移（BRET）。量子点是微小的荧光半导体纳米晶体，可以产生具有限定发射波长的光谱并用于多重检测。在量子点-BRET过程中，量子点可以通过来自发光蛋白的共振能量转移过程发光。这项研究将开发一系列基于QD-BRET平台的纳米传感器，以检测和成像三种重要的蛋白酶，这些蛋白酶高度参与肿瘤转移：MMP-2，明胶酶A，负责降解IV型胶原蛋白，细胞外基质的主要成分; MMP-7，基质溶解素，具有广泛蛋白水解活性的最小成员;和尿激酶型纤溶酶原激活剂（uPA），涉及MMP的激活。有三个具体目标：1）建立QD-RBET检测平台，用于生物样品中MMP-2、MMP-7和uPA的多重分析（在细胞培养基和小鼠血清中）; 2）对小鼠模型中异种移植肿瘤中的MMP-2、MMP-7和uPA活性进行多重成像; 3）观察小分子干扰RNA对uPA、MMP-2和MMP-7的抑制作用，探讨三种蛋白酶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标记物的早期检测和肿瘤活性和功能的体内实时监测都将是非常宝贵的。在此制定的战略也可以扩展到许多其他目标。对活体肿瘤和正常组织蛋白水解活性差异的深入了解将促进我们对癌症转移的理解，并有助于开发抗转移治疗。 公共卫生相关性：这项研究旨在开发新型纳米技术，以检测和成像促进肿瘤从原发部位迁移到远程器官的关键酶。这种新的纳米技术将允许高度灵敏地检测生物样品中的这些酶分子，以帮助早期发现癌症。深入了解活体肿瘤组织和正常组织中酶活性的差异，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.