Abstract：
A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.
摘要：
用于将治疗剂递送至细胞和组织的流行策略是将治疗剂封装在细胞经由内吞作用内化的颗粒内。通常使用流式细胞术和Western印迹分析大量研究通过内吞作用吸收颗粒的功效，并使用共聚焦显微镜进行确认。然而,这些技术没有揭示颗粒内化的详细动力学,以及许多类型颗粒的固有异质性如何影响它们的内吞摄取.为了解决这些差距，在这里，我们提出了一种基于活细胞成像的方法，该方法利用全内反射荧光显微镜来跟踪大量单个颗粒的平行摄取，因为它们与细胞内吞机制相互作用。为了分析结果数据，我们采用开源跟踪算法与自定义数据过滤器相结合。该分析揭示了颗粒和内吞结构之间的动态相互作用，这决定了粒子摄取的概率。特别是,我们的方法可用于检查颗粒物理性质的变化(大小，瞄准,刚性),以及粒子群体内的异质性，影响内吞摄取。这些数据影响颗粒的设计,以更有选择性和有效地将治疗剂递送至细胞。
