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

项目摘要 这种更新的应用程序建立在我们的发现，纳米粒子为基础的结构的形状-我们 定义为结构价-在保持纳米结构的靶向配体特征方面起关键作用 即使被蛋白质冠包被。蛋白质冠的形成及其对工程理化性质的影响 纳米结构的性质有些争议，但通常被接受为影响细胞和组织 靶向特异性和下游生物效应。使用单粒子，活细胞成像研究，我们 发现纳米结构集合体的蛋白质冠组成被单颗粒取代， 形状效应与它们的球形对应物相比，具有分支结构的纳米结构保留了它们的 配体壳靶向细胞膜上和细胞内以及细胞区室的能力。统计学 不同形状的纳米结构与细胞相互作用的时间分辨动力学的显着差异 组分之间的差异不能归因于蛋白质冠组成的非常小的变化。 我们的目标是确定分支纳米结构如何保持其靶向功能的机制 然后调节单颗粒蛋白质冠的空间组织， 光在活细胞相互作用。我们将调整分支纳米结构的纳米尺度特征，包括 总体大小、分支长度和尖端曲率半径，与膜受体二聚化相当 和曲率诱导的内体信号传导，并将在单粒子水平上成像相互作用。具体 目的包括（1）了解纳米结构设计和光学刺激如何在蛋白质的表面操纵蛋白质冠， 单颗粒水平;（2）研究膜靶向分支纳米构建物的结构多价性 （3）使用分支纳米构建体形状改善细胞内靶向和递送。 这项工作有望通过单纳米粒子的交叉对精密纳米医学产生重大影响 对生物反应机制的深入了解。我们解决纳米颗粒-细胞相互作用的能力 在相关的时间和长度尺度上进行受体识别和内化将有益于蛋白质 二聚化和纳米曲率效应，加深对内吞作用和免疫调节作用的理解。 反应-疫苗设计和药物输送进步的关键过程。

PROJECT SUMMARY This renewal application builds on our discovery that the shape of nanoparticle-based constructs—what we define as structural valency—plays a key role in preserving the targeting ligand characteristics of nanoconstructs even when coated by a protein corona. Protein corona formation and its impact on engineered physicochemical properties of nanoconstructs has been somewhat controversial but is generally accepted to affect cell and tissue targeting specificity and downstream biological effects. Using single-particle, live-cell imaging investigations, we found that the protein corona composition of an ensemble of nanoconstructs was superseded by single-particle shape effects. Compared to their spherical counterparts, nanoconstructs with branched structures retained their ligand shell targeting capabilities both on cell membranes and within cells and cellular compartments. Statistically significant differences in the time-resolved dynamics of differently shaped nanoconstructs interacting with cellular components cannot be attributed to very minimal variations in protein corona composition. We aim to determine the mechanism for how branched nanoconstructs maintain their targeting functionality in biological environments and then to modulate the spatial organization of the single-particle protein corona with light during live-cell interactions. We will tune the nanoscale features of the branched nanoconstructs, including overall size, branch length, and tip radii of curvature, to be commensurate with membrane receptor dimerization and curvature-induced endosomal signaling and will image interactions at the single-particle level. The specific aims include (1) Understand how nanoconstruct design and optical stimuli can manipulate the protein corona at the single-particle level; (2) Investigate structural multivalency of membrane-targeting branched nanoconstructs on receptor dimerization; (3) Improve intracellular targeting and delivery using branched nanoconstruct shapes. This work is expected to be of high impact for precision nanomedicine by the intersection of single-nanoparticle advances with mechanistic insight into biological responses. Our ability to resolve nanoparticle-cell interactions at relevant time and length scales for receptor recognition and internalization will benefit knowledge in protein dimerization and nanoscale curvature effects and deepen understanding of endocytosis and immunomodulatory responses—key processes for advancements in vaccine design and drug delivery.