Comparison of two in vitro systems to assess cellular effects of nanoparticles-containing aerosols

Abstract

Inhalation treatment with nanoparticle containing aerosols appears a promising new therapeutic option but new formulations have to be assessed for efficacy and toxicity. We evaluated the utility of a VITROCELL®6 PT-CF+PARI LC SPRINT® Baby Nebulizer (PARI BOY) system compared with a conventional MicroSprayer. A549 cells were cultured in the air-liquid interface, exposed to nanoparticle aerosols and characterized by measurement of transepithelial electrical resistance and staining for tight junction proteins. Deposition and distribution rates of polystyrene particles and of carbon nanotubes on the cells were assessed. In addition, cytotoxicity of aerosols containing polystyrene particles was compared with cytotoxicity of polystyrene particles in suspension tested in submersed cultures. Exposure by itself in both exposure systems did not damage the cells. Deposition rates of aerosolized polystyrene particles were about 700 times and that of carbon nanotubes about 4 times higher in the MicroSprayer than in the VITROCELL®6 PT-CF system. Cytotoxicity of amine-functionalized polystyrene nanoparticles was significantly higher when applied as an aerosol on cell cultured in air-liquid interface culture compared with nanoparticle suspensions tested in submersed culture. The higher cytotoxicity of aerosolized nanoparticles underscores the importance of relevant exposure systems.

Fig. 1

Exposure systems. (a): The main compounds of the VITROCELL®6 PT/PARI BOY exposure system are indicated: PARI BOY® SX compressor connected by tubes to the PARI BOY LC SPRINT® Baby nebulizer produces the aerosol. The flow of the aerosol from the nebulizer via a glass tube to the three individual compartments of the exposure module until the collection of aerosol not delivered to the compartments in a plastic vial is indicated. Airflow is regulated by a vacuum pump (not seen) that aspirates air from each of the three compartments of the exposure module through plastic tubes. For temperature maintenance at 37 °C a water bath connected to the exposure unit by plastic tubing, is used. (b): The tube of the MicroSprayer IA-1C aerosolizer is attached with a clamp to a tripod and the position of the tip is fixated at a distance of 11 cm from the rim of the exposure well. The long flexible tube allows the activation of the syringe to generate the aerosol without displacing the aerosolizer tip.

Fig. 2

Physiological and morphological characterization of not exposed A549 cells cultured in air–liquid interface (ALI) culture. (a): Development of the transepithelial electrical resistance (TEER) in A549 cells cultured in transwells. Means ± SD of n = 5 experiments are given. (b–e): Immunocytochemical detection of tight junction proteins after 7 days in ALI culture: Claudin-1 (red, b), ZO-1 (green, c) and E-cadherin (red, d) is seen between A549 cells cultured on transwells. Co-labeling of ZO-1 with E-cadherin (e) shows that the proteins are localized in different membrane areas. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Distribution of fluorescently labeled (red) carboxyl-functionalized polystyrene particles (FluoSpheres, FS) on A549 cells 24 h after exposure in the VITROCELL®6 PT-CF/PARI BOY system. Cells are stained by phalloidin (green) and counterstained with Hoechst 33342 (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Deposition rate (% of nanoparticles in the respective compartment referred to total aerosolized nanoparticles) and distribution rate (% of nanoparticles in the respective compartment normalized to the total amount deposited) of fluorescein, fluorescently-labeled 20 nm and larger (40–200 nm) carboxyl-functionalized polystyrene particles (FluoSpheres, FS, a) and carbon nanotubes of <8 nm (CNT8), 20–30 nm (CNT20) and >50 nm (CNT50, b) in the VITROCELL®6 PT-CF/PARI BOY system. Data are presented as means ± SD of n = 3 experiments. The three chambers of the VITROCELL®6 PT-CF exposure unit are termed as 1st, 2nd, and 3rd compartment. Significant differences in the deposition and distribution of the respective compartment to the 1st compartment are indicated by an asterisk and significant differences to the respective values in the 2nd compartment by a hash (p < 0.05).

Fig. 5

Cytotoxicity of amine-functionalized polystyrene particles in A549 cells assessed by the MTS assay. The particles were tested either in suspension on submersed culture (liquid culture, LCC) or as aerosol on ALI cultures with the MicroSprayer. Concentrations are indicated as μg/cm² and are based on a deposition rate of 29% as determined in previous experiments. Viability of solvent exposed cells is set as 100%. Data are presented as means ± SD of n = 4 experiments and significant differences (p < 0.05) are indicated by an asterisk.