Characterization of Nanomaterial Dispersion in Solution Prior to In Vitro Exposure Using Dynamic Light Scattering Technique

Abstract

The need to characterize nanoparticles in solution before assessing the in vitro toxicity is a high priority. Particle size, size distribution, particle morphology, particle composition, surface area, surface chemistry, and particle reactivity in solution are important factors which need to be defined to accurately assess nanoparticle toxicity. Currently, there are no well-defined techniques for characterization of wet nanomaterials in aqueous or biological solutions. Previously reported nanoparticle characterization techniques in aqueous or biological solutions have consisted of the use of ultra-high illumination light microscopy and disc centrifuge sedimentation; however, these techniques are limited by the measurement size range. The current study focuses on characterizing a wide range of nanomaterials using dynamic light scattering (DLS) and transmission electron microscopy, including metals, metal oxides, and carbon-based materials, in water and cell culture media, with and without serum. Cell viability and cell morphology studies were conducted in conjunction with DLS experiments to evaluate toxicological effects from observed agglomeration changes in the presence or absence of serum in cell culture media. Observations of material-specific surface properties were also recorded. It was also necessary to characterize the impact of sonication, which is implemented to aid in particle dispersion and solution mixture. Additionally, a stock solution of nanomaterials used for toxicology studies was analyzed for changes in agglomeration and zeta potential of the material over time. In summary, our results demonstrate that many metal and metal oxide nanomaterials agglomerate in solution and that depending upon the solution particle agglomeration is either agitated or mitigated. Corresponding toxicity data revealed that the addition of serum to cell culture media can, in some cases, have a significant effect on particle toxicity possibly due to changes in agglomeration or surface chemistry. It was also observed that sonication slightly reduces agglomeration and has minimal effect on particle surface charge. Finally, the stock solution experienced significant changes in particle agglomeration and surface charge over time.

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

“…Brown et al for example found that nanorods and -fibres agglomerate more easily than spheres and Limbach et al saw a higher agglomeration rate for smaller NP in comparison to their larger counterparts at similar mass doses, which was explained by the higher number density 172,173 . This is of course also related to the concentration, which is another important factor as it is known that NP tend to agglomerate at higher concentrations 116 175,176 . Still, it can be concluded that NP agglomeration can either be promoted or mitigated depending on the medium composition 176 .…”

“…This is of course also related to the concentration, which is another important factor as it is known that NP tend to agglomerate at higher concentrations 116 175,176 . Still, it can be concluded that NP agglomeration can either be promoted or mitigated depending on the medium composition 176 .…”

Ecotoxicity test methods for engineered nanomaterials: Practical experiences and recommendations from the bench

“…The optimal sonication time depends on the suspended ENM concentration, but it is usually less than 1 h [35]. In addition, sonication can introduce artefacts in toxicity studies because it fragments multiwalled carbon nanotubes [36] and possibly alters the coatings on ENMs [37] or increases reactive oxygen species (ROS) production (see later discussion). Sonication settings and procedures should therefore be reported carefully at all times.…”

Antimicrobial properties of nano-silver: A cautionary approach to ionic interference

“…A deeper investigation reveals that many of these reports merely append a simplistic disk diffusion assay or a variation of this assay to essentially a material synthesis report [26,10]. Often such studies fail to take account of the physicochemical properties of the nanoparticle which have been crucially important in helping to standardise approaches for the assessment of nanoparticle toxicity and are likewise expected to contribute significantly to understanding the antimicrobial properties of nanomaterials [19,28]. In addition, limited statistical analysis is often carried out in the aforementioned work with the inhibition zone being taken as conclusive evidence that the nanomaterial under study is antimicrobial with no regard to impurities, particle coatings, competing processes or known mechanisms of interaction [25,1,17].…”

“…In the disk diffusion assay a micro-organism is classed as sensitive or resistant to a material based on the radius of the zone of inhibition which forms around a small filter paper disk containing the test material. In comparison to antibiotics, the properties of nanomaterials are less consistent, with significant variations between batches in terms of particle size distribution, bioactivity, purity and surface chemistry [28,19]. Furthermore nano-metals such as nano-silver are often insoluble in aqueous solution and have a tendency to agglomerate in certain environments such as nutrient broth [19] making them unsuitable for many assays other than the disk diffusion assay.…”