title: A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles.
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 mug/cm(2). 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 mug/cm(2)) 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 mug/cm(2 )ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Lung Biology and Disease, Ingolstaedter Landstrasse 1, D-85758 Neuherberg, Germany.
authors.forename: Anke Gabriele
authors.initials: AG
authors.lastname: Lenz
authors.forename: Erwin
authors.initials: E
authors.lastname: Karg
authors.forename: Bernd
authors.initials: B
authors.lastname: Lentner
authors.forename: Vlad
authors.initials: V
authors.lastname: Dittrich
authors.forename: Christina
authors.initials: C
authors.lastname: Brandenberger
authors.forename: Barbara
authors.initials: B
authors.lastname: Rothen-Rutishauser
authors.forename: Holger
authors.initials: H
authors.lastname: Schulz
authors.forename: George A
authors.initials: GA
authors.lastname: Ferron
authors.forename: Otmar
authors.initials: O
authors.lastname: Schmid