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）已经显示出将治疗剂和诊断剂递送到体内疾病部位的巨大潜力。1 -6传统上，研究一直集中在能够在患病组织内被动积累的NPs的设计上，7-10或通过采用肿瘤相关细胞上过表达受体的展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于与肿瘤组织相关的酶信号的形态和纳米级结构的物理变化来积累NPs的主动靶向方法。14 -17在这些活性纳米载体中，其动态刺激响应行为是其功能的关键要素。然而，对这种形态转变的基本机制知之甚少，主要是由于缺乏足够的技术来实时观察纳米级动态系统。6，18这种缺乏理解极大地限制了下一代活性纳米载体的发展，其中优化依赖于理解复杂液体环境中的基本作用和响应机制。拟议研究的长期目标是发展有关动态刺激响应材料和工艺的纳米级机制和动力学的基础知识。原位液体细胞透射电子显微镜（LCTEM）技术18，19将被开发和建立作为独特的工具，用于成像有机纳米结构在生理液体。拟议的工作代表了利用TEM对液体中的这种材料进行成像的第一次努力，这可以为LCTEM常规用于表征包括病毒和细胞成分在内的动态生物纳米结构的未来奠定基础。在这里，将研究两种不同的刺激响应NP系统，一种酶响应14 -17和一种pH响应，这两种系统在液体-颗粒界面处发生刺激诱导的反应后都经历形态学转变。将构建新型超薄石墨烯液体电池20、21用于LCTEM表征，克服与这些水合有机结构的图像对比度和检测22 -25相关的问题（目标1）。初始LCTEM实验将使用预混合溶液在反应进程的各个阶段对合成和反应后形态进行成像，为所涉及的动态转化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用于石墨烯液体电池 观察完整的刺激响应行为，从最初的反应事件到最终的平衡形态（目的2）。将通过视觉和多目标跟踪计算方法26 -28分析获得的原位LCTEM视频，以阐明转化的机制和动力学。研究结果将揭示快速转型的限制性和促进性步骤。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性质，以创建具有定制的刺激响应转化行为的新纳米载体，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.