Super-Localization Ultrasound Imaging with Targeted Laser-activated Nanodetectors

 描述(申请人提供)：虽然癌症是由单个细胞的突变驱动的，但肿瘤是复杂的环境，更像器官而不是简单的细胞团。最近的研究表明，由于肿瘤具有不同的遗传特征以及肿瘤实质细胞与周围基质细胞之间的相互作用，肿瘤内的微环境具有极大的异质性。这种异质性与肿瘤的恶性及其对治疗的抵抗力有关。尽管异质肿瘤微环境很重要，但目前还没有工具可以充分显示其在体内的动力学。这项提议旨在开发一种新的成像技术-超定位超声成像-依靠高度动态的相变纳米颗粒来创建肿瘤内分子表达的高分辨率图谱。这种相变造影剂被称为激光激活纳米探测器(LANS)，由脂单分子层组成，用包裹的染料稳定液体全氟己烷核心。在脉冲激光照射下，平台发生快速汽化，形成瞬时微泡，导致超声成像信号增加。当全氟己烷冷却到沸点(56°C)以下时，熔体重新凝结成稳定的液滴状态。再凝结时间是一个随机过程，它取决于液滴大小、入射超声能量和局部组织环境。因此，高帧频超声成像能够捕获单个再凝结事件。连续超声图像之间的差异揭示了成像系统仅对单个激活陆地的反应。在将此响应与成像系统的点扩展函数进行拟合后，可以以比系统的衍射/带宽限制分辨率高得多的精度来定位地面的准确位置。在将LAND与分子特异性抗体结合后，可以获得组织中分子表达的高分辨率地图。这项应用侧重于LAND的开发和优化以及相关的超声成像技术。针对表皮生长因子受体的分子靶向将通过与单抗的定向结合来实现。将合成这些粒子，并用动态光散射、紫外-可见光谱和透射电子显微镜对其进行充分表征。同时，将开发和优化定制的超声成像序列，以高灵敏度检测单个LAND的再凝结。分辨率的整体提高将完全体现在模仿组织的幻影中。最后，通过异种移植小鼠癌症模型进行的活体研究将被用于研究该技术的安全性、向肿瘤输送LAND、分子特异性以及超定位超声成像检测异质肿瘤中微小空间差异的能力。最终的结果将是一个多功能的框架，可以用于实时和在体研究肿瘤中各种分子的表达。这具有广泛的意义，从加深我们对癌症进展的理解到在临床上对治疗反应的个性化预测。

 DESCRIPTION (provided by applicant): Although cancer is driven by mutations in individual cells, tumors are complex environments, resembling organs more than simple clusters of cells. Recent studies have shown that the microenvironment within tumors is wildly heterogeneous, owing to tumors with diverse genetic profiles as well as interactions between the tumor parenchymal cells and the surrounding stromal cells. This heterogeneity is linked to the malignancy of tumors and their resistance to therapy. Despite the importance of the heterogeneous tumor microenvironment, there are no tools that can adequately visualize its dynamics in vivo. This proposal seeks to develop a new imaging technology - super-localization ultrasound imaging - which relies on highly dynamic phase-change nanoparticles to create a high resolution map of molecular expressions within tumors. The phase-change contrast agents, termed laser-activated nanodetectors (LANDs), consist of a lipid monolayer stabilizing a liquid perfluorohexane core with an encapsulated dye. Upon irradiation with a pulsed laser, the LANDs undergo a rapid vaporization and a transient microbubble is formed, resulting in an increased signal in ultrasound imaging. As the perfluorohexane cools below its boiling point (56 °C) the LANDs recondense into their stable droplet state. The time of recondensation is a stochastic process, and it depends on the droplet size, incident interrogating ultrasound energy, and the local tissue environment. Therefore, high frame rate ultrasound imaging is able to capture single recondensation events. The difference between successive ultrasound images reveals the response of the imaging system to only a single activated LAND. After fitting this response to the point spread function of the imaging system, the exact position of the LAND can be localized with much greater precision than the system's diffraction/bandwidth limited resolution. After conjugating the LANDs with molecular-specific antibodies, a high-resolution map of the molecular expressions in tissue can be obtained. This application focuses on the development and optimization of the LANDs as well as the associated ultrasound imaging techniques. Molecular targeting to the epidermal growth factor receptor will be achieved via a directional conjugation to a monoclonal antibody. The particles will be synthesized and fully characterized with dynamic light scattering, ultraviolet-visible spectrophotometry, and transmission electron microscopy. Simultaneously, custom ultrasound imaging sequences will be developed and optimized to detect the recondensation of individual LANDs with high sensitivity. The overall improvement in resolution will be fully characterized in tissue-mimicking phantoms. Finally, in vivo studies with a xenograft mouse model of cancer will be used to study the safety of the technique, the delivery of LANDs to the tumor, the molecular specificity, and the ability of super-localization ultrasound imaging to detect small spatial variations in a heterogeneous tumor. The end result will be a versatile framework that can be used to study a wide variety of molecular expressions in tumors in real-time and in vivo. This has broad implications ranging from furthering our understanding of the progression of cancer to personalized prediction of treatment response in the clinic.