Continuous Probing of Nanoconstruct-Cell Interactions at Biologically Relevant Time Scales

项目总结 这项提议旨在开发活细胞、多通道成像工具，这种工具可以连续、 实时-纳米颗粒与细胞组件的相互作用。我们将关注几个不同的时间窗口， 其中早期时间段将监测纳米粒子-细胞膜的结合和摄取，而稍后时间段将跟踪 内体堆积和逃逸。能够在时间和空间上解析颗粒大小/形状和 配基密度影响3D中的相互作用是确定结构-活性-关系和机制的关键 在活细胞中的作用。要表征功能纳米颗粒与不同细胞结构的相互作用，请访问 在生理相关的时代，我们提出了设计一种集成了光学显微镜的多通道光学显微镜。 分离器和定制的活细胞成像室。可以在不同的通道中同时采集图像 染料标记细胞器和染料标记配体在颗粒上的荧光及其区别 整个细胞、细胞组件和纳米颗粒核心的干涉对比(DIC)信号。的相关性 结构和功能图像提供了一个强大的窗口，了解局部纳米结构相互作用如何 调节一种生物反应。对于模型系统，我们将比较金纳米结构和寡核苷酸 具有靶向性(DNA适配子)或非靶向性(SiRNA)的配位体外壳。纳米颗粒形状使 一个独特的手柄，用于探测细胞内颗粒相互作用的旋转和方向。这项工作可以弥补差距。 在理解纳米结构在细胞内的行为以及如何完整和呈现 配体壳层中的寡核苷酸影响靶向和其他过程，如内体逃逸，这是 对评估治疗效果至关重要。

PROJECT SUMMARY This proposal aims to develop live-cell, multi-channel imaging tools that can visualize—continuously, and in real time—nanoparticle interactions with cellular components. We will focus on several different time windows, where early time periods will monitor nanoparticle-cell membrane binding and uptake and later times will track endosomal accumulation and escape. The ability to resolve temporally and spatially how particle size/shape and ligand density affects interactions in 3D is critical for determining structure-activity-relationships and mechanism of action in live cells. To characterize interactions of functional nanoparticles with different cellular structures at physiologically relevant times, we propose to design a multi-channel optical microscope integrated with an opto- splitter and custom live-cell imaging chamber. Simultaneous images can be acquired in different channels of the fluorescence of dye-labeled organelles and dye-labeled ligands on the particles as well as differential interference contrast (DIC) signals of whole cells, cellular components, and nanoparticle cores. Correlation of structural and functional images provides a powerful window into how local nanoconstruct interactions can mediate a biological response. For model systems, we will compare gold nanoconstructs with oligonucleotide ligand shells of both targeting (DNA aptamers) or non-targeting (siRNA) properties. Nanoparticle shape enables a unique handle to probe rotation and orientation of intracellular particle interactions. This work can bridge a gap in understanding the behavior of nanoconstructs intracellularly and how the integrity and presentation of oligonucleotides in ligand shells affects targeting and other processes such as endosomal escape, which is critical to assess therapeutic efficacy.