10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
Abstract View
Optimization of DAVID Cell Exposure System for Toxicity Analysis of Nanoparticles at the Air-Liquid Interface
TREVOR TILLY, Ryan Ward, Jiva Luthra, Sarah Robinson, Arantzazu Eiguren Fernandez, Saber Hussain, Tara Sabo-Attwood, John Lednicky, Chang Yu Wu, University of Florida
Abstract Number: 1336 Working Group: Aerosols and Health - Connecting the Dots
Abstract Understanding the health effects associated with the inhalation of nanoparticles is critical for determining the risk they pose during occupational and environmental exposures. The many different types and variations of nanoparticles has made evaluating their toxicity burdensome by in vivo methods alone, and resulted in the development of many different in vitro toxicity analyses. Despite this development, the current existing methods of in vitro have drawbacks when evaluating occupational and environmental exposures of nanoparticles resulting from their fundamental properties. For instance, the process of collecting nanoparticles from the environment on filters and then later extraction in media for toxicity analysis in the laboratory can greatly alter the properties of the particles directly after emission, and subsequently alter their toxicity outcome. To remedy the incorporation of these particle transformations that can result from sample collection, air-liquid interface (ALI) exposure systems have been developed to deliver nanoparticles from aerosols directly to cells. However, deposition of nanoparticles on cells at the ALI may be skewed to a certain size range of particles because the aerosol methods of thermophoresis and electrostatic precipitation often utilized to increase deposition efficiency in these systems are dependent on particle charge and size. A new cell exposure system known as DAVID, Dosimetric Aerosol in Vitro Inhalation Device, was designed to improve the delivery of a broad range of nanoparticles through increased inertial force accomplished by amplifying particles as small as 10 nm to a uniform size of 3 µm by condensing water vapor onto the particle. With this technology, evaluation of the toxicity of nanoparticles was achievable with short exposure times by delivering a concentration of particles that met or exceeded the lowest observed adverse effect level. Custom cell culture supports were fabricated by 3D printing, functionalized by oxygen plasma corona for cell attachment, and sterilized with ethanol and UV. With the current setup, four cell membranes cultured at the ALI can be exposed simultaneously so that statistical analysis can be performed within each exposure trial. First, the delivery of clean air to the cell surface was optimized in DAVID to ensure that the flow rate would not cause adverse effect to the cells by drying them out. A flow rate of 2.1 L/minute/cm2 was selected based on sampling recommendations, but resulted in a reduction of cell viability by 25% of control. Therefore, the flow rate was reduced to 1.4 L/minute/cm2 and was found to maintain greater than 97% viability of the cells. The deposition of nanoparticles in DAVID was calibrated and determined to increase linearly with time from an uranine aerosol generated by a Collison nebulizer with a mean particle size of approximately 58 nm. The raw fluorescence of the collected uranine droplets on the membranes was measured and related to a calibration curve to determine the deposition mass concentration. Nanoparticle concentration in the nebulizer and dilution flow rate was directly related to the mass deposition of particles in DAVID, which resulted in a mass deposition ranging from 177 ng/cm2 to 2.93 µg/cm2 for exposures up to 20 minutes. Post optimization, the cells were exposed to CeO nanoparticles with mean diameter of 39.5 nm generated by the Collison nebulizer in the laboratory, and the delivered dose was assessed by inductively-coupled plasma mass spectrometry. The viability of the cells after exposure was assessed by lactate dehydrogenase assay (LDH) and related to the deposited dose of particles on the cells. Future studies to determine mechanistic nanoparticle-cell interactions at the ALI are the next step for the DAVID exposure system.