Typical in vitro assays used for high throughput toxicological screening and measuring nano-bio interactions are conducted by pipetting suspensions of engineered nanomaterials (ENMs) dispersed in nutrient-rich culture media directly onto cells. In order to achieve fairly monodisperse and stable suspensions of small agglomerates, ultrasonic energy is usually applied to break apart large agglomerates that can form upon suspension in liquid. Lack of standardized protocols and methods for delivering sonication energy can introduce variability in the ENM suspension properties (e.g. agglomerate size, polydispersity, suspension stability over time), and holds significant implications for in vitro dosimetry, toxicity, and other nano-bio interactions. Careful assessment of particle transformations during dispersion preparation and sonication is therefore critical for accurate interpretation of in vitro toxicity studies. In this short communication, the difficulties of preparing stable suspensions of rapidly settling ENMs are presented. Furthermore, methods to optimize the delivery of the critical sonication energy required to break large agglomerates and prepare stable, fairly monodispersed suspensions of fast settling ENMs are presented. A methodology for the efficient delivery of sonication energy in a discrete manner is presented and validated using various rapidly agglomerating and settling ENMs. The implications of continuous vs. discrete sonication on average hydrodynamic diameter, and polydispersity was also assessed for both fast and slow settling ENMs. For the rapidly agglomerating and settling ENMs (Ag15%/SiO2, Ag and CeO2), the proposed discrete sonication achieved a significant reduction in the agglomerate diameter and polydispersity. In contrast, the relatively slow agglomerating and settling Fe2O3 suspension did not exhibit statistically significant differences in average hydrodynamic diameter or polydispersity between the continuous and discrete sonication approaches. Our results highlight the importance of using the proposed material-specific discrete sonication method to effectively deliver the critical sonication energy necessary to reproducibly achieve stable and fairly monodispersed suspensions that are suitable for in vitro toxicity testing.

中文翻译：

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有效地将超声能量传递到快速沉降和凝聚的纳米材料悬浮液中以进行细胞研究：对稳定性、粒子动力学、剂量测定和毒性的影响


用于高通量毒理学筛选和测量纳米生物相互作用的典型体外测定是通过将​​分散在营养丰富的培养基中的工程纳米材料 (ENM) 的悬浮液直接移液到细胞上来进行的。为了实现小团聚体的相当单分散和稳定的悬浮液，通常应用超声波能量来分解在液体中悬浮时形成的大团聚体。缺乏用于传递超声能量的标准化协议和方法可能会导致 ENM 悬浮液特性的变化（例如附聚物大小、多分散性、悬浮液随时间的稳定性），并对体外剂量测定、毒性和其他纳米生物相互作用产生重大影响。因此，在分散体制备和超声处理过程中仔细评估颗粒转变对于准确解释体外毒性研究至关重要。在这篇简短的交流中，介绍了制备快速沉降 ENM 的稳定悬浮液的困难。此外，还提出了优化破碎大团聚体和制备稳定、相当单分散的快速沉降 ENM 悬浮液所需的临界超声能量传递的方法。提出了一种以离散方式有效传递超声能量的方法，并使用各种快速凝聚和沉降的 ENM 进行了验证。还评估了快速和慢速沉降 ENM 的连续超声处理与离散超声处理对平均流体动力学直径和多分散性的影响。对于快速团聚和沉降的 ENM（Ag15%/SiO2、Ag 和 CeO2），所提出的离散超声处理实现了团聚直径和多分散性的显着降低。 相比之下，相对较慢的团聚和沉降 Fe2O3 悬浮液在连续和离散超声处理方法之间的平均流体动力学直径或多分散性没有表现出统计学上的显着差异。我们的结果强调了使用所提出的特定于材料的离散超声处理方法来有效地提供可重复地实现适合体外毒性测试的稳定且相当单分散的悬浮液所需的临界超声能量的重要性。