In situ Liquid Cell Transmission Electron Microscopy (LCTEM) for Observing Tumor Responsive Nanocarriers

 描述(申请人提供)：纳米颗粒(NPs)已经显示出在活体中将治疗和诊断传递到疾病部位的巨大潜力1-6传统上，研究集中于能够在病变组织内被动积累的NPs的设计，7-10或通过使用肿瘤相关细胞上过表达的受体的显示配体的主动靶向方法。2，3，6，11-13相比之下，Gianneschi实验室的重点一直是通过响应与肿瘤组织相关的酶信号的物理变化来积聚NPs的主动靶向方法。14-17在这些活性纳米载体中，它们的动态刺激反应行为是其功能的关键要素。然而，人们对这种形态转变背后的基本机制知之甚少，这主要是因为缺乏足够的技术来实时观察纳米级动态系统。6，18这种缺乏了解的情况极大地限制了下一代活性纳米载体的发展，在这种情况下，优化依赖于了解复杂液体环境中的作用和响应的基本机制。拟议研究的长期目标是发展关于动态刺激响应材料和过程的纳米机制和动力学的基础知识。原位液体细胞透射式电子显微镜(LCTEM)技术18、19将被开发和确立为在生理液体中成像有机纳米结构的独特工具。这项拟议的工作是首次尝试利用透射电子显微镜在液体中对这种类型的材料进行成像，这可以为未来LCTEM常规用于表征包括病毒和细胞组件在内的动态生物纳米结构奠定基础。在这里，我们将研究两个不同的刺激响应NP系统，一个是酶响应的14-17，一个是pH响应的，这两个系统都在刺激诱导的液-颗粒界面发生反应后发生形态变化。新型超薄石墨烯液晶盒20、21将用于LCTEM表征，克服与图像对比度和检测这些水合有机结构22-25相关的问题(目标1)。最初的LCTEM实验将使用预混合溶液在反应进程的不同阶段成像合成和反应后的形态，为了解所涉及的动态转变过程提供第一条线索。最终，使用酶或酸性溶液的液体流动能力将被用于石墨烯液体电池。 观察完整的刺激反应行为，从最初的反应事件到最终的平衡形态(目标2)。获得的原位LCTEM视频将通过多目标跟踪计算方法26-28进行可视化分析，以阐明转变的机制和动力学。结果将揭示快速转型的限制和支持步骤。有了这些知识，合成化学家可以智能地修改胶束构建块的化学成分，以创建具有量身定制的刺激响应转换行为的新纳米载体，以改善体内行为。

 DESCRIPTION (provided by applicant):Nanoparticles (NPs) have shown tremendous potential for the delivery of therapeutics and diagnostics to sites of disease in vivo.1-6 Traditionally, research has been focused on the design of NPs capable of passive accumulation within diseased tissue,7-10 or via an active targeting method employing displayed ligands for overexpressed receptors on tumor associated cells.2,3,6,11-13 By contrast, the focus of the Gianneschi lab has been on active targeting methods for accumulating NPs through physical changes in morphology and nanoscale structure in response to enzymatic signals associated with tumor tissue.14-17 In these active nanocarriers, it is their dynamic stimuli-responsive behavior that is the critical element in their functionality. However, very little is known about te fundamental mechanisms that underlie morphology transitions of this kind, primarily due to a lack of adequate techniques to observe nanoscale dynamic systems in real-time.6,18 This lack of understanding greatly limits the development of next-generation active nanocarriers where optimization is dependent on understanding basic mechanisms of action and response in complex liquid milieu. The long-term objective of the proposed research is to develop fundamental knowledge about the nanoscale mechanisms and kinetics of dynamic stimuli-responsive materials and processes. In situ liquid cell transmission electron microscopy (LCTEM) techniques18,19 will be developed and established as unique tools for imaging organic nanostructures in physiological liquids. The proposed work represent the first efforts to utilize TEM for imaging this type of material in liquid, which can lay the foundation for a future n which LCTEM is routinely used to characterize dynamic biological nanostructures including viruses and cellular components. Here, two different stimuli-responsive NP systems will be studied, one enzyme-responsive14-17 and one pH-responsive, both of which undergo morphological transformations after stimuli-induced reactions occur at the liquid-particle interface. Novel ultra-thin graphene liquid cells20,21 will be constructed for LCTEM characterization, overcoming issues related to image contrast and detection22-25 of these hydrated organic structures (Aim 1). Initial LCTEM experiments will image the synthesized and post-reaction morphologies at various stages of reaction progression using pre-mixed solutions, providing the first clues into the dynamic transformation processes involved. Ultimately, liquid-flow capabilities, using enzyme or acidic solution, will be employed with the graphene liquid cells to observe the complete stimuli-responsive behaviors, from the initial reaction events through to their final equilibrium morphologies (Aim 2). The acquired in situ LCTEM videos will be analyzed visually and by multi-target tracking computational methods26-28 to elucidate the mechanisms and kinetics of transformation. The results will expose the limiting and enabling steps for rapid transformation. With this knowledge, synthetic chemists can intelligently modify the chemistry of the micelle building blocks to create new nanocarriers with tailored stimuli-responsive transformation behaviors for improved in vivo behavior.