基于信号放大的铁蛋白纳米粒子检测系统用于肺部微小转移瘤的活体成像研究

Detection of lung metastases by current preclinical or clinical imaging techniques has substantial limitations. Although the rapid development of engineered nanoparticles with integration of different functions shows a great promise for the diagnosis and treatment of disease, nanoparticles for in vivo targeted imaging of lung metastases have not been available in clinic. Low sensitivity is one of major problems, especially for nuclear magnetic resonance (NMR) imaging. Thus, a pair of ferritin-based nanoparticles with signal amplification is developed to improve the sensitivity. First, signaling nanoparticles with targeting ability to lung metastases are designed bearing a great number of signaling molecules, while detecting nanoparticles with fluorescence/NMR activity can specially bind to the signaling molecules on the surface of the signaling nanoparticles. With this pair of nanoparticles, signals for the imaging can be amplified based on the fact that a large quantity of the detecting nanoparticles are accumulated in the lung metastases. This is because those tiny tumors could have been labeled by huge amount of signaling molecules, which are conjugated to signaling nanoparticles. In this research, both signaling and detecting nanoparticles are generated from multifunctional ferritin H-chain nanoparticles by using genetic method and chemical modifications. Next, with both cell and blood vessel models we can study the key factors contributed to the signal amplification and optimize the characteristics of both nanoparticles in vitro for the best outcome. Then, to address the possibility of those nanoparticles for in vivo imaging of tiny metastases, the sensitivity and the limit of detection for imaging cells are determined in vitro. After that, both signaling and detecting nanoparticles are used for in vivo near infrared/NMR imaging of lung metastases. With animal models we are able to determine the amplification effect introduced by signaling nanoparticles, and evaluate the pairs of nanopartices for detecting the metastasis lesions smaller than 3 mm in diameter. All these work not only show a significant improvement of the sensitivity for in vivo imaging of lung metastases, but also provide a good method for applying system nanoparticles in diagnosing tiny tumors in lung tissue.

目前缺乏肺部微小转移瘤的特异性成像常规检测技术。尽管纳米材料的制备与多功能化技术的极大发展推动了它们在癌症诊断与治疗方面的应用，但相关纳米材料用于肺部微小转移瘤的诊断成像尚未临床化。特异性成像的灵敏度不够高是其中的关键原因之一。为此，本课题拟通过基因工程、蛋白表达与纯化以及化学偶联技术构建一对"信号/检测纳米粒子"，发展一种以提高检测灵敏度为目的信号放大检测技术。在体内信号纳米粒子能召集大量的检测纳米粒子富集于肺部微小转移瘤是该技术的核心。研究将以肿瘤细胞等为模型，静态条件下研究检测系统信号放大的效应与机制；并采用微流控芯片模拟肿瘤血管环境，优化系统的放大效应；以及确定体外成像的灵敏度。同时，还将构建乳腺癌肺转移动物模型，研究该系统的体内放大效应和对肺微小转移瘤的活体成像。这些研究不仅为肺部微小转移瘤的诊断提供一种高灵敏度的成像方法，也为探索系统纳米生物医学在疾病诊断与治疗提供策略和方法。

目前缺乏肺部微小转移瘤的特异性成像常规检测技术。尽管纳米材料的制备与多功能化技术的极大发展推动了它们在癌症诊断与治疗方面的应用，但相关纳米材料用于肺部微小转移瘤的诊断成像尚未临床化。特异性成像的灵敏度不够高是其中的关键原因之一。为此，本课题拟通过基因工程、蛋白表达与纯化以及化学偶联技术构建一对“信号/检测纳米粒子”，发展一种以提高检测灵敏度为目的信号放大检测技术。在体内信号纳米粒子能召集大量的检测纳米粒子富集于肺部微小转移瘤是该技术的核心。本项目首先探索了多功能化策略构建蛋白类纳米粒子，这些研究不仅提供了多种铁蛋白多功能化修饰的方法，同时还提供了一种通过分子动力学方法考察自组装体蛋白表面修饰对结构稳定性研究的思路，拓展了自组装蛋白在纳米生物医学领域应用与发展。同时还构建了多种多功能化铁蛋白纳米粒子，并考察了它们在纳米生物医学领域的应用。随后，以肿瘤细胞等为模型，发展了两对“信号/检测纳米粒子”，详细研究检测系统信号放大的效应与机制。结果显示我们所构建的其中一对信号纳米粒子Biotin-EGF/HSA-FTH1和检测纳米粒子mSA-FTH1/FTH1-FITC能够有效地放大其检测的荧光信号，能有效提高检测的灵敏度，并且特别适合受体低表达的细胞的诊断成像。而荧光显微镜则进一步显示了这些纳米粒子的相互结合是发生在细胞表面。最后，构建了乳腺癌动物模型，研究该系统的体内放大效应和肿瘤的活体成像，与单一靶向EGF/HSA-FTH1纳米粒子相比，我们所构建的纳米检测系统信号提高147%。这些研究不仅为肿瘤的诊断提供一种高灵敏度的成像方法，也为探索系统纳米生物医学在疾病诊断与治疗提供策略和方法。