Nanopore Force Spectroscopy and Sorting of Vesicles at Nanoscale

项目摘要 纳米尺度囊泡形成细胞器的结构框架，例如溶酶体、内体、外泌体， 内吞囊泡和外吞囊泡，以及病毒的脂质包膜。这些生理或病理的 纳米载体是自然界的分子传递系统，因此代表了开发 用于制药应用的新型药物/基因递送系统。囊泡的一个重要方面是， 机械性能使它们能够完成看似完全不同的任务：1-变形并与目标融合 2-在动态生物学中保持物理完整性而不破裂， 环境.例如，纯的合成囊泡（即脂质体）不具有足够的机械性能。 完整性，以有效地承受生物环境中存在的恶劣扰动（例如，大的波动 在静态压力下），但通过更复杂的结构特征，如膜蛋白和 蛋白质-脂质复合物，可以提供结构完整性，同时保持变形和融合的能力。 靶向膜。因此，研究囊泡的力学性质，并了解其机制， 的增强，以及在纳米尺度上的囊泡力学可溶性效应器的影响，是很大的 对基本目的和应用目的都有重要意义。它不仅能帮助我们理解 生物运输现象，而且还可以导致生物启发的药物/基因递送系统的新解决方案。 脂质体的力谱是了解囊泡力学性质最直接的方法， 然而，利用现有技术，在纳米级脂质体上进行力谱分析是非常具有挑战性的。 在溶液中。目前最先进的技术，原子力光谱（AFM）是昂贵的和时间- 消耗量大、通量低，并且需要训练有素的操作员和复杂的样品制备。 此外，目前还没有可以基于囊泡的机械性质来分离和分选囊泡的方法。 性能，限制了我们直接比较机械性能与功能特性的能力。 在这个项目中，我们将开发一种基于纳米孔的力谱方法，克服了 原子力显微镜，表征纳米脂质体的机械性能，并可以根据其分类脂质体。 力学性能其两个具体目标是目标1：检测和测量各种机械 在固态纳米孔中使用电阻脉冲传感的纳米级囊泡的特性，以及目标2：开发 一种自动反馈控制系统，可以根据纳米囊泡的机械特性对其进行分类。 所提出的纳米孔力谱可用于表征天然纳米材料的机械性能。 发生的纳米囊泡，如病毒，外来体等。此外，自动反馈控制系统 将被开发，可以分离出所需的机械性能（如刚度）的混合样品， 具有不同性质的囊泡群体。这样的平台可用于分选异质生物样品。 样品的基础上，他们的机械行为，并研究在纳米生物力学的功能作用。

PROJECT SUMMARY Nanoscale vesicles form the structural framework of organelles such as lysosomes, endosomes, exosomes, endocytic and exocytic vesicles, as well as the lipid envelope for viruses. These physiological or pathological nanocarriers are nature’s delivery systems for molecules and therefore represent prototypes for developing novel drug/gene delivery systems for pharmaceutical applications. An important aspect of vesicles is that their mechanical properties allow them to achieve seemingly diametrical tasks: 1- to deform and merge with target membranes to deliver their cargos, and 2- to maintain physical integrity without rupturing in dynamic biological environments. Pure synthetic vesicles (i.e. liposomes), for example, do not possess sufficient mechanical integrity to effectively withstand harsh perturbations present in biological environments (e.g. large fluctuations in static pressure), but reinforcement by more complex structural features, such as membrane proteins and protein-lipid complexes, can provide structural integrity, while maintaining the ability to deform and to fuse with target membranes. Hence, studying the mechanical properties of vesicles, and understanding the mechanisms of reinforcement, as well as the effect of soluble effectors on vesicles’ mechanics at the nanoscale, is of great significance for both fundamental and applied purposes. Not only can it help us understand the fundamental biological transport phenomena, but also can lead to new solutions for bio-inspired drug/gene delivery systems. Force spectroscopy of liposomes is the most direct way to understand mechanical properties of vesicles, however, with the current technologies it is very challenging to do force spectroscopy on nanoscale liposomes in solution. The current state-of-the-art technique, atomic force spectroscopy (AFM) is expensive and time- consuming, is low-throughput, and requires highly-trained operators and complex sample preparation. Furthermore, there is currently no method that can separate and sort vesicles based on their mechanical properties, limiting our ability to directly compare mechanical properties with functional characteristics. In this project we will develop a nanopore based force spectroscopy method, that overcomes limitations of AFM, to characterize the mechanical properties of nanoscale liposomes and can sort liposomes based on their mechanical properties. Two specific aims of this are Aim 1: to detect and measure varied mechanical properties of nanoscale vesicles using resistive pulse sensing in solid-state nanopores, and Aim 2: to develop an automated feedback-controlled system that can sort nanovesicles based on their mechanical properties. The proposed nanopore force spectroscopy can be used to characterize mechanical properties of naturally- occurring nanovesicals such as viruses, exosomes, etc. In addition, an automated feedback-controlled system will be developed that can separate samples of desired mechanical properties (e.g. rigidity) out of a mixed population of vesicles with varied properties. Such a platform can be used to sort heterogeneous biological samples based on their mechanical behavior and study the functional role of biomechanics at the nanoscale.