Toxicological assessment of inhaled nanoparticles: role of in vivo, ex vivo, in vitro, and in silico studies

Abstract

The alveolar epithelium of the lung is by far the most permeable epithelial barrier of the human body. The risk for adverse effects by inhaled nanoparticles (NPs) depends on their hazard (negative action on cells and organism) and on exposure (concentration in the inhaled air and pattern of deposition in the lung). With the development of advanced in vitro models, not only in vivo, but also cellular studies can be used for toxicological testing. Advanced in vitro studies use combinations of cells cultured in the air-liquid interface. These cultures are useful for particle uptake and mechanistic studies. Whole-body, nose-only, and lung-only exposures of animals could help to determine retention of NPs in the body. Both approaches also have their limitations; cellular studies cannot mimic the entire organism and data obtained by inhalation exposure of rodents have limitations due to differences in the respiratory system from that of humans. Simulation programs for lung deposition in humans could help to determine the relevance of the biological findings. Combination of biological data generated in different biological models and in silico modeling appears suitable for a realistic estimation of potential risks by inhalation exposure to NPs.

Figure 1.

Barriers for particle uptake by the respiratory system. Surfaces of larger (conducting) airways are mainly covered by bronchial epithelial cells with cilia (BE) and mucus (blue) producing goblet cells (GC). In bronchioli, bronchial epithelial cells and mucus producing cells (Clara cells, C) are found. All epithelial cells reside on a basement membrane (BM). The air-blood barrier at the alveolus consists of alveolar epithelial cells type I (AT-I) and surfactant-producing AT-II cells. Alveolar macrophages (M) migrate on top of the alveolar epithelial cell layer. On the other side of the basement membrane endothelial cells (EC) of capillaries are located.

Figure 2.

Fate of inhaled nanoparticles in conducting airways (bronchial epithelium) and alveoli. Particles can be either absorbed through the bronchial epithelium and enter systemic circulation or removed from the bronchial epithelium by mucociliary clearance (MC) and then absorbed in the gastrointestinal tract (GIT). Absorption pathway: black arrows; metabolization and excretion: blue arrows.

Figure 3.

Exposure of alveolar cells in submerged culture and exposed to nanoparticle suspensions (a), and cultured in air-liquid interface exposed to nanoparticle-loaded aerosol (b). (a) Alveolar cells (AC) cultured in submersed culture usually do not differentiate and lack mucus or surfactant. NPs suspended in medium often form aggregates; (b) (co-culture shown): Alveolar cells cultured on transwells in the air-liquid interface produce surfactant (blue). NPs in aerosols usually form smaller aggregates than nanoparticles in suspensions. For further refinement of the model, co-culture with macrophages (M) on top of the epithelial cells (b) can be used.

Figure 4.

Set-up for aerosol-exposure at the air-liquid interface based on the VitroCell^® (Vitrocell Systems GmbH, Waldkirch, Germany) exposure system (a) and by manually generated aerosols (MicroSprayer^®, b). (a) Cells are cultured on transwells and exposed in the compartments of the exposure unit, thermo stabilized by a water bath. Airflow generated by a vacuum pump provides a steady flow of the particle-loaded aerosol, generated in the particle generator (PARI BOY LC Sprint, PARI GmbH, Starnberg, Germany), over the cells. Part of the aerosol that passes the glass tube, is collected at the end of the glass tube and used for particle analysis in the aerosol. Particle deposition on cells is quantified in one compartment of the exposure unit; (b) Cells cultured on transwells are exposed in a separate exposure plate to one to three puffs of manually generated aerosol.

Figure 5.

Exposure of rodents to aerosols. (a) nose-only exposure. The restraint (R) prevents loss of aerosol by leakage around the animal. The small opening at the bottom allows temperature regulation of the animal through the tail; Intratracheal instillation (b) and oropharyngeal aspiration (c) uses commercial or self-designed syringes for manual application of aerosol.

Figure 6.

Particle parameters important for deposition in the whole lung. Large particles are subjected to inertial impactation, preferentially in large airways, smaller particles deposit by gravitational sedimentation, and small particles in the alveoli by diffusion. Electrostatic deposition is seen for charged particles and interception for fiber-shaped particles.