(137c) Generation of Complex in Vitro Systems for Improved Evaluation of Nanoparticle-Induced Bioresponses

Cellular systems are required to test the safety of newly developed materials, chemicals, and biological-based products to ensure their safety.  Both in vitro and in vivo methodologies are being utilized for evaluation, however, limited correlation exists between these models. This discrepancy is due to the drastic, fundamental differences between basic cell culture conditions and fully functioning organisms. One way to overcome this limitation is through the development of enhanced in vitro environments that retain the advantages of cellular systems but more accurately mimic physiology.  Studies are beginning to emerge that attempt to bridge the gap between these models by improving upon standard in vitro techniques through the addition of physiologically relevant variables.  Alterations currently being explored include, the formation of cellular co-cultures, the use of physiological fluid instead of cell culture media, generation of a 3-dimensional growth substrate, and the introduction of shear stress through dynamic flow. However, to date, the simultaneous combination of more than one of these advancements has yet to be executed.

In the work presented here, we successfully developed and implemented two enhanced in vitro microenvironment that more closely mimicked an in vivo system, while retaining the advantages of cell-based techniques. These systems, which consisted of multiple modulations to standard in vitro techniques, were employed for the evaluation of nanoparticle (NP) bioeffects and the nano-bio interface.  NPs were specifically chosen owing to their unique physicochemical properties that distinguish them from chemical species, such as their insoluble nature, ability to undergo ionic dissolution, agglomeration tendencies, and NP sedimentation effects. In particular, silver and gold NPs (AgNPs and AuNPs) were selected, as they are the most prevalent and highly utilized composition in the consumer market. As inhalation is a primary route of exposure for NPs, that was our target cellular system.  

In the first microenvironment, alveolar epithelial cells (A549) were exposed to AgNPs and AuNPs in artificial alveolar fluid (AAF) and under dynamic flow.  Dynamic flow was established through the use of a peristaltic pump and was set to a volumetric flowrate that produce physiologically accurate linear velocities. The NPs demonstrated significant behavioral alterations in the microenvironment, specifically targeting NP agglomeration, spectral profiles, and kinetic rates of dissolution.  For example, following dispersion in AAF, aggregate sizes of AuNPs increased over 2-fold from a traditional media environment.  Beyond NP effects, the cellular response was greatly varied following the addition of AAF or shear stress.  In agreement with greater agglomeration and decreased ionic dissolution, the stress and cytotoxic responses to AgNPs were significantly reduced in AAF.  While the influence of dynamic flow on the NPs was negligible, it greatly modified both A549 morphology and the nano-bio interface.  In a flowing system, NP deposition was reduced over 20%, with TEM imaging confirming modified endocytosis mechanisms. 

 The goal of the second constructed microenvironment was to allow for augmented evaluation of biological responses following NP exposure; therefore the system was expanded to include a co-culture consisting of A549 alveolar epithelial cells and U937 macrophages.  The effectiveness of this co-culture model was first evaluated by confirming that the U937 cells were preferentially engulfing the sub-toxic dose of AgNPs.  Next, dynamic flow was introduced and again, we observed a dramatic drop in NP deposition: approximately 40% less than the static environment. Interesting, for the co-culture model, the shear stress induced by dynamic flow did translate to greater biological stress, as evaluated through LDH release and ROS production.  However, the inflammatory response, assessed through secretion of target cytokines, following AgNP exposure was decreased under flow, correlating with the lower degree of NP deposition.  Taken together, these results support the emerging idea that numerous environmental variables can influence NP properties and behaviors, with the potential to affect downstream bioeffects and overall safety evaluations. To that end, the utilization of a complex and physiologically relevant models is necessary to accurately and effectively evaluate the safety and performance of NPs in nano-based applications.