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

项目摘要 这项提案旨在开发活细胞，多通道成像工具，可以可视化-连续，并在 真实的时间-纳米粒子与细胞成分的相互作用。我们将关注几个不同的时间窗口， 早期将监测纳米颗粒与细胞膜的结合和吸收， 内体积累和逃逸。能够在时间和空间上解析颗粒大小/形状以及 配体密度影响3D中的相互作用对于确定结构-活性关系和机制至关重要 在活细胞中的作用。为了表征功能纳米颗粒与不同细胞结构的相互作用， 生理相关的时间，我们建议设计一个多通道光学显微镜集成与光， 分离器和定制活细胞成像室。可以在不同的通道中同时采集图像， 荧光染料标记的细胞器和染料标记的配体上的颗粒，以及微分 全细胞、细胞组分和纳米颗粒核心的干涉对比（DIC）信号。相关性 结构和功能图像提供了一个强有力的窗口，以了解局部纳米结构相互作用如何 介导生物反应。对于模型系统，我们将比较金纳米结构与寡核苷酸 具有靶向（DNA适体）或非靶向（siRNA）性质的配体壳。纳米颗粒形状使 一种独特的手柄，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 在理解细胞内纳米结构的行为以及纳米结构的完整性和呈现方式方面， 配体壳中的寡核苷酸影响靶向和其他过程，如内体逃逸， 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.