Institute of Life Sciences, Swansea University Medical School , Singleton Park, Swansea, SA2 8PP, U.K.
Centre for Nanohealth, Swansea University College of Engineering , Fabian Way, Crymlyn Burrows, Swansea, SA1 8EN, U.K.
ACS Nano. 2017 Dec 26;11(12):11986-12000. doi: 10.1021/acsnano.7b03708. Epub 2017 Nov 1.
Understanding the delivered cellular dose of nanoparticles is imperative in nanomedicine and nanosafety, yet is known to be extremely complex because of multiple interactions between nanoparticles, their environment, and the cells. Here, we use 3-D reconstruction of agglomerates preserved by cryogenic snapshot sampling and imaged by electron microscopy to quantify the "bioavailable dose" that is presented at the cell surface and formed by the process of individual nanoparticle sequestration into agglomerates in the exposure media. Critically, using 20 and 40 nm carboxylated polystyrene-latex and 16 and 85 nm silicon dioxide nanoparticles, we show that abrupt, dose-dependent "tipping points" in agglomeration state can arise, subsequently affecting cellular delivery and increasing toxicity. These changes are triggered by shifts in the ratio of the total nanoparticle surface area to biomolecule abundance, with the switch to a highly agglomerated state effectively changing the test article midassay, challenging the dose-response paradigm for nanosafety experiments. By characterizing nanoparticle numbers per agglomerate, we show these tipping points can lead to the formation of extreme agglomeration states whereby 90% of an administered dose is contained and delivered to the cells by just the top 2% of the largest agglomerates. We thus demonstrate precise definition, description, and comparison of the nanoparticle dose formed in different experimental environments and show that this description is critical to understanding cellular delivery and toxicity. We further empirically "stress-test" the commonly used dynamic light scattering approach, establishing its limitations to present an analysis strategy that significantly improves the usefulness of this popular nanoparticle characterization technique.
了解纳米颗粒的细胞内递呈剂量在纳米医学和纳米安全性中至关重要，但由于纳米颗粒与其环境以及细胞之间的多种相互作用，其已知极其复杂。在这里，我们使用低温快照采样保存的聚集体的 3D 重建，并通过电子显微镜成像，来量化在细胞表面呈现并由单个纳米颗粒在暴露介质中被聚集体隔离的过程形成的“生物有效剂量”。至关重要的是，我们使用 20 和 40nm 羧基化聚苯乙烯-乳胶以及 16 和 85nm 二氧化硅纳米颗粒，表明团聚状态会突然出现剂量依赖性的“临界点”，继而影响细胞摄取并增加毒性。这些变化是由纳米颗粒总表面积与生物分子丰度之比的变化引发的，高度团聚状态的转变实际上会在实验中途改变测试物质，对纳米安全性实验的剂量反应范式提出挑战。通过对每个聚集体中的纳米颗粒数量进行表征，我们表明这些临界点会导致极端团聚状态的形成，其中 90%的给药剂量仅由最大聚集体的前 2%来承载和递送至细胞。因此，我们精确地定义、描述和比较了在不同实验环境中形成的纳米颗粒剂量，并表明这种描述对于理解细胞摄取和毒性至关重要。我们进一步对常用的动态光散射方法进行了实证“压力测试”，确定了其局限性，并提出了一种分析策略，可显著提高这种流行的纳米颗粒特征分析技术的有用性。
