Effective delivery of sonication energy to fast settling and agglomerating nanomaterial suspensions for cellular studies: Implications for stability, particle kinetics, dosimetry and toxicity

Abstract

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.

Keywords: Engineered nanomaterials; agglomeration; dispersion preparation; nanotoxicology; sonication.

Figure 1. Overview of continuous vs. discrete sonication

a) Discrete sonication is the process whereby sonication energy is delivered in short intervals, interspersed with periods of vortexing to ensure mixing and a homogenous distribution of particles throughout the sample. This process is then repeated until the delivered energy reaches the DSE_cr. Continuous sonication involves sonicating the suspension, without stopping, until the DSE_cr is reached. Rapidly agglomerating materials will settle out of suspension, sonication energy will not effectively be delivered resulting in an unstable suspension of large agglomerates. Therefore such materials require the discrete sonication approach to effectively deliver the DSE_cr and achieve small uniform agglomerates. b) Slow settling materials, for which the formed agglomerates are relatively easy to break as indicated by very low DSEcr values (blue squares), require fewer DSE intervals to sufficiently break apart the large agglomerates.

Figure 2. ENM-specific DSEcr and Dispersion Stability

a) Determination of DSE_cr for 10nm Ag15%/SiO₂. b) average hydrodynamic diameter of suspension sonicated below DSE_cr; at time 0 and 24 hours post sonication; c) average hydrodynamic diameter of suspension sonicated at the DSE_cr; at time 0 and 24 hours post sonication; d) average hydrodynamic diameter of suspensions sonicated above DSE_cr; at time 0 and 24 hours post sonication.