10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Real-time Dosimetry for In-vitro Toxicological Studies of Engineered Nanoparticles (ENPs) at the Air-liquid Interface Using a Quartz Crystal Microbalance (QCM)
Yaobo Ding, Patrick Weindl, Clara Wimmer, Paula Mayer, Tobias Krebs, OTMAR SCHMID, Helmholtz Zentrum München, Germany
Abstract Number: 1182 Working Group: Aerosols and Health - Connecting the Dots
Abstract Inhalation of engineered nanoparticles (ENPs), despite their unique physico-chemical properties compared to conventional materials, has been found to bear potential adverse effects on human health. In inhalation toxicology studies, direct air-to-cell exposure of ENP aerosols as possible with air-liquid interface (ALI) cell cultures provides a more physiologically-relevant platform to investigate their toxic effects, as compared to the traditional submerged cell culture systems. Moreover, the submerged cell exposures suffer from inaccurate control of the cell-delivered dose, which can be resolved by incorporating real-time dose measurement devices in ALI exposure systems.
In this study, we characterized the performances of the VITROCELL® CLOUD system (6- & 12-well model; VITROCELL Systems, Germany) for aerosolized ENP delivery to cells as well as the integrated QCM for measurement of the cell-delivered ENP dose. The system is equipped with a vibrating membrane nebulizer (Aeroneb Pro, Aerogen, Ireland) which generates aerosol droplets from a liquid suspension containing ENPs. As described by Lenz et al (2014) in the VITROCELL CLOUD system the aerosol droplets form a dense cloud, which utilizes cloud dynamics to rapidly (within 3 min) fill the exposure chamber, to uniformly mix the aerosol by vortex formation and to finally sediment the ENPs spatially uniform onto the transwell inserts containing the cells. A QCM is incorporated to monitor the cell-deposited dose.
A fluorescein solution was nebulized to investigate the uniformity and efficiency of aerosol deposition onto the cells cultured in transwell inserts as well as on the QCM. For the CLOUD 6 the deposition efficiency was 70.3%±1.9% and 63.5%±4.4% for inserts and the QCM, respectively, indicating no statistically significant difference. This was also the case for the CLOUD 12, but with slightly higher deposition efficiency (76-81%). These values are only weakly depended on the type of Aeroneb mesh nebulizers used. Out of six different nebulizers with varied output rates and droplet sizes, five resulted in the same deposition efficiency. Insert-to-insert variability was 4.4% and 6.0% for CLOUD 6 and CLOUD 12, respectively, within the range of nebulized volumes of ENP suspensions (50-400 µl). The QCM accuracy was 3.4% and 3.8% for CLOUD 6 and 12, respectively, as determined by using quantitative fluorescence spectroscopy on fluorescein as reference dosimetry method. The lower detection limit of the QCM was 500 and 170 ng/cm2 for CLOUD 6 and CLOUD 12, respectively, which is above the onset dose for most types of ENPs. Finally, human lung epithelial cells (A549) were exposed to ZnO (JRC-NM110) nanoparticle aerosols and a dose-response relation was established from toxicological endpoints including cell viability (IC50=ca. 0.5 cm2/cm2=4.2 µg/cm2, membrane integrity and pro-inflammatory level.
Our results show that the VITROCELL-CLOUD systems can be used to deliver high dose rates of aerosolized ENPs (<1000 ng/cm2/min) uniformly (<6% insert-insert variability) and efficiently to cells cultured at the air-liquid interface. Dose-response relationships can be readily determined with an integrated QCM providing real-time information on the cell-delivered ENP dose.