Research

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

Anke Gabriele Lenz¹ , Erwin Karg¹ , Bernd Lentner¹ , Vlad Dittrich¹ , Christina Brandenberger² , Barbara Rothen-Rutishauser² , Holger Schulz¹ , George A Ferron¹ and Otmar Schmid¹

¹  Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Lung Biology and Disease, Ingolstaedter Landstrasse 1, D-85758 Neuherberg, Germany

²  University of Bern, Institute of Anatomy, Division of Histology, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland

author email corresponding author email

Particle and Fibre Toxicology 2009, 6:32doi:10.1186/1743-8977-6-32

Published:	16 December 2009

Abstract

Background

Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.

Results

A novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm². The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.

Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm²) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm² ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.

Conclusion

The ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.