Implementation of physiological fluids to provide insight into the characterization, fate, and biological interactions of silver nanoparticles.

Silver nanoparticles (AgNPs) are being increasingly utilized in consumer and medical applications. However, there remains conflicting reports on their safety, which are evaluated through a combination of in vitro and in vivo exposure models. These discrepancies may arise, in part, due to the inherent differences between cell-based and animal systems. It is well established that nanotoxicological effects are highly dependent on the unique physicochemical properties and behavior of the particle set, including size, surface chemistry, agglomeration, and ionic dissolution. However, recent studies have identified that these properties vary as a function of exposure environment; providing a rationale for the contradictory results between in vitro and in vivo assessments. Artificial physiological fluids are emerging as a powerful tool as they allow for the characterization of NPs in an environment which they would likely encounter in vivo, in addition to having the experimental advantages of flexibility and consistency. Here, we demonstrated that the utilization of artificial fluids provided a mechanism to assess AgNP behavior and induced bioresponses in environments that they would likely encounter in vivo. AgNPs were introduced within an alveolar-based exposure model, which included alveolar epithelial (A549) cells incubated within artificial alveolar fluid (AF). Additionally, the particles underwent extensive characterization within both AF and lysosomal fluid, which the AgNPs would encounter following cellular internalization. Following incubation in physiological environments AgNP properties were significantly modified versus a traditional media environment, including alterations to both extent of agglomeration and rate of ionic dissolution. Moreover, when A549s were exposed to AgNPs in AF, the cells displayed lower cytotoxicity and stress rates, corresponding to a fluid-dependent drop in silver ion production. This work highlights the need for enhanced in vitro models that more closely mimic in vivo exposure environments in order to capture true NP behaviors and cellular interactions.

Exposure to metal oxide nanoparticles in physiological fluid induced synergistic biological effects in a keratinocyte model.

Nanoparticles (NPs) possess distinctive physicochemical properties that in addition to differentiating them from their bulk counterparts can induce negative cellular consequences. Standard in vitro systems have served as the primary model for NP safety evaluations, but suffer from a lack physiological relevance. One way to overcome this limitation and evaluate NP characteristics under more accurate conditions is through the use of artificial physiological fluids, which mimic the composition of in vivo environments. In this study, we identified that copper oxide (CuO) and titanium dioxide (TiO2) NPs displayed modified behavior when dispersed in artificial interstitial fluid (IF) versus traditional media, including extensive agglomeration and increased particle deposition. When keratinocyte cells underwent CuO NP exposure, synergistic stress and toxicity responses occurred within an IF environment, correlating with augmented particle deposition. However, following IF incubation alone or concurrently with TiO2 NPs, which are not innately toxic, no combinatorial responses were identified. These results indicate that synergistic outcomes arise when toxic NPs undergo fluid-induced alterations to key physicochemical properties and behaviors. This study highlights the necessity of carrying out NP characterization and safety assessments in physiologically-representative environments; as altered behavior patterns have the potential to induce bioresponses not identified within traditional models.

The role of biological fluid and dynamic flow in the behavior and cellular interactions of gold nanoparticles.

BACKGROUND: Due to their distinctive physicochemical properties, nanoparticles (NPs) have proven to be extremely advantageous for product and application development, but are also capable of inducing detrimental outcomes in biological systems. Standard in vitro methodologies are currently the primary means for evaluating NP safety, as vast quantities of particles exist that require appraisal. However, cell-based models are plagued by the fact that they are not representative of complex physiological systems. The need for a more accurate exposure model is highlighted by the fact that NP behavior and subsequent bioresponses are highly dependent upon their surroundings. Therefore, standard in vitro models will likely produce inaccurate NP behavioral analyses and erroneous safety results. As such, the goal of this study was to develop an enhanced in vitro model for NP evaluation that retained the advantages of cell culture, but implemented the key physiological variables of accurate biological fluid and dynamic flow. RESULTS: In this study, a cellular microenvironment was modeled and created after an inhalation exposure scenario. This system comprised of A549 lung epithelial cells, artificial alveolar fluid (AAF), and biologically accurate dynamic flow. Under the influence of microenvironment variables, tannic acid coated gold NPs (AuNPs) displayed modulated physicochemical characteristics, including increased agglomeration, disruption of the spectral signature, and decreased rate of ionic dissolution. Furthermore, AuNP deposition efficiency, internalization patterns, and the nano-cellular interface varied as a function of fluid composition and flow condition. AAF incubation simultaneously influenced both AuNPs and cellular behavior, through excessive NP agglomeration and alteration to A549 morphology. Dynamic flow targeted the nano-cellular interface, with differential responses including modified deposition, internalization patterns, and cellular elongation. Lastly, the biocompatibility of the system was verified to ensure cellular health following AAF exposure and fluid dynamics. CONCLUSIONS: This study confirmed the feasibility of improving standard in vitro models through the incorporation of physiological variables. Utilization of this enhanced system demonstrated that to elucidate true NP behavior and accurately gauge their cellular interactions, assessments should be carried out in a more complex and relevant biological exposure model.

Dynamic characteristics of silver nanoparticles in physiological fluids: toxicological implications.

The field of nanotoxicology has made tremendous progress identifying novel and potentially adverse biological effects following nanomaterial (NM) exposure. However, one facet yet to be satisfactorily explored is how a physiological environment modifies NM physicochemical properties, thus introducing novel complexities associated with solid phase material exposures. In this study, artificial alveolar, lysosomal, and interstitial fluids were used to identify environmental-specific modulations to the properties and behavior of hydrocarbon-coated (Ag-HC) and polysaccharide-coated (Ag-PS) silver NMs. As inhalation is a common route of exposure, an alveolar macrophage cell model with deposition dosages representing approximately 2.5 months and 10 years of occupational exposure (0.5 and 25 ng/mL, respectively) were employed. Following dispersion in the artificial fluids, the Ag-HC and Ag-PS NMs demonstrated significant alterations to morphology, aggregation patterns, and particle reactivity. However, the Ag-PS also demonstrated a loss of particle coating, which elicited increased cytotoxicity, phagocytosis, and inflammation not associated with the original Ag-PS. This study demonstrated that in a physiological system NMs undergo considerable modulation, introducing a scenario where the toxicity of NMs may increase over time due to internal bioconditions. These findings highlight the critical influence that the dynamic and insoluble nature of NMs have on bioeffects and the importance of characterizing this behavior.