由“蛋白酶响应性”操控的近红外/磁共振双显影智能自组装微囊

As one kind of enzymes that hold the capability of catalyzing the hydrolysis of peptide amide bonds, proteases are known to be overexpressed in tumors and its expression level has been correlated with metastatic potential. Therefore the design and fabrication of protease-responsive imaging probes are of great importance in the early diagnosis of tumor. However, there was few report related to the development of protease-responsive imaging probe in both near infrared fluorescent imaging (NIFI) and magnetic resonance imaging (MRI). We developed a self-assembly approach which had been verified effective in fabricating MRI probes, our studies identified that the microcapsules displayed an interesting variable MRI T2 signal which decreased sharply upon the disassembly of the microcapsules. Inspired by this property of the microcapsules, herein we propose an approach to fabricate the NIFI/MRI dual imaging probes which are protease-responsive in both NIFI and MRI. The reseach in this proposal includes three parts as follows. (1) The general idea for developing the NIFI/MRI dual imaging probes is to construct microcapsules from magnetic nanoparticles and polypeptide which is responsive to a specific protease, and embedding a combination of near infrared fluorochromes and spectrally matched "quencher" molecules into microcapsules during their fabrication. In this native state the near infrared microcapsules are designed to be maximally quenched, which upon hydrolysis of polypeptide and consequent disassembly of microcapsules become brightly fluorescent to detect the protease. Combining the decrease of MRI signal and the increase of NIFI signal upon the disassembly of microcapsules, the microcapsules are anticipated to be developed as the novel dual imaging probe which is responsive to protease in both MRI and NIFI. (2) Based on the successful fabrication of these dual imaging probes, we are going to study the in vitro protease-responsive imaging capability of these NIFI/MRI dual imaging probes, as well as the relationship between the structure of microcapsules and their properties. (3) Finally, several typical cells will be chosen to study the cytotoxicity and cell uptake of microcapsules, and on this basis, for the determination of tissue signal intensities after intravenous injection of microcapsule probes, appropriate animal models will be chosen to study in vivo imaging capability of the microcapsule probes. Basically, this study will develop a self-assembly approach to construct novel protease-responsive imaging probes which are responsive to protease in both NIFI and MRI, and study the imaging capability of the probes after elucidating the relationship between the structure and properties of microcapsules.

作为一类可选择催化肽键水解的酶，蛋白酶与多种肿瘤细胞的生长与转移密切相关，因而发展蛋白酶响应的显影探针对肿瘤早期诊断具有重要意义。但迄今，相关研究并不多见，而在近红外(NIFI)和磁共振显影(MRI)中兼具蛋白酶响应性的探针更未见报道。我们前期已成功由"无模板自组装"得到MR显影微囊，并发现其MRI信号随微囊"解组装"而突变。受此启发，本研究拟用如下思路构筑蛋白酶响应的双显影探针，并系统研究其性能。(1)在磁性微囊制备中引入蛋白酶敏感的多肽，并将近红外荧光探针及其淬灭剂共同包封于微囊内，在蛋白酶触发的微囊"解组装"中，荧光探针-淬灭剂间距离的增加将导致探针淬灭-解淬灭状态的突变，结合此间微囊MRI显影的降低，得到在NIFI/MRI中兼具蛋白酶敏感性的微囊。(2)研究模拟体液中微囊的蛋白酶响应性，并探讨其结构-性能关系。(3)开展微囊在细胞及小动物显影中的性能研究，为其生物医学应用奠定基础。

作为一类可选择催化肽键水解的酶，蛋白酶与多种肿瘤细胞的生长与转移密切相关，因而发展蛋白酶响应的显影探针对肿瘤早期诊断具有重要意义。但迄今，相关研究并不多见，而在近红外(NIFI)和磁共振显影(MRI)中兼具蛋白酶响应性的探针更未见报道。我们前期已成功由“无模板自组装”得到MR显影微囊，并发现其MRI信号随微囊“解组装”而突变。基于如上研究背景和研究基础，本研究采用如下思路构筑了蛋白酶响应的双显影探针，并对其性能和生物医学显影应用进行了系统研究。本项目具体完成了如下研究内容：(1)在磁性微囊制备中引入了蛋白酶敏感的多肽，并将近红外荧光探针包封于微囊内，利用荧光分子间的非辐射能量转移使得其在自组装微囊中处于淬灭状态。在蛋白酶触发的微囊“解组装”中，荧光探针间距离的增加导致探针淬灭—解淬灭状态的突变，结合此间微囊MRI显影的降低，从而得到在NIFI/MRI中兼具蛋白酶敏感性的微囊。(2)选用生物相容性大分子、ZIF系列金属有机框架对制备的组装体进行了表面修饰，并对表面修饰-复合自组装微囊环境响应性能间的关系进行了一系列研究。(3)研究了模拟体液中微囊的蛋白酶响应性，并系统探讨了这类微囊的结构—性能关系。(4)开展微囊在细胞及小动物显影中的性能研究，为其生物医学应用奠定了良好基础。本研究的意义在于利用多层级组装的思路，直接由纳米粒子-多肽间的多层级自组装成功制备了具有良好蛋白酶响应性的自组装微囊，并为将这一思路拓展至多种肿瘤诊断显影探针的制备做出了具有普适性意义的尝试。

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