The effect of titanium dioxide nanoparticles on pulmonary surfactant function and ultrastructure

Abstract

Background: Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO(2)) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO(2) effects on surfactant ultrastructure were visualized.

Methods: A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 microg/ml) of TiO(2) NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope.

Results: TiO(2) NSP, but not MSP, induced a surfactant dysfunction. For TiO(2) NSP, adsorption surface tension (gammaads) increased in a dose-dependent manner from 28.2 + or - 2.3 mN/m to 33.2 + or - 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (gammamin) slightly increased from 4.8 + or - 0.5 mN/m up to 8.4 + or - 1.3 mN/m (p < 0.01) at high TiO(2) NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both gammaads (63.6 + or - 0.4 mN/m) and gammamin (21.1 + or - 0.4 mN/m). Interestingly, TiO(2) NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae.

Conclusion: TiO(2) nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.

Figure 1

Surface activity evaluated with the pulsating bubble surfactometer. A) Adsorption surface tension (γ_ads) after incubation with TiO₂ nanosized particles (NSP) at a static bubble condition. B) Influence of TiO₂ microsized particles (MSP) on γ_ads C) Influence of TiO₂ NSP on surface tension at minimal bubble size (γ_min) during pulsation D) γ_min after incubation with TiO₂ MSP. Values are given as means of at least 4 experiments ± SEM. ** indicates p values < 0.01 compared with the control at 0 μg/ml particle concentration.

Figure 2

Surface activity evaluated with the pulsating bubble surfactometer following 8 hour rotation at 0.43 Hz. A) Influence of TiO₂ NSP on adsorption surface tension (γ_ads) at a static bubble condition. B) Influence of TiO₂ MSP on γ_ads C) Influence of TiO₂ NSP on surface tension at minimal bubble size (γ_min) during pulsation D) TiO₂ MSP effect on γ_min. Values are given as means ± SEM of at least 4 experiments. ** indicates p values < 0.01; *** indicates p values < 0.001; both compared with the rotated 0 μg/ml particle concentration (grey columns). CO/white columns - control surfactant which was placed for 8 hours in an incubator without rotation.

Figure 3

Representative transmission electron microscope pictures of the surfactant ultrastructure. A) and B) untreated control surfactant. C) and D) porcine surfactant after addition of 100 μg/ml TiO₂ nanosized particles. Red circles show small particle aggregates. E) Control surfactant after 8 hours rotation at 0.43 Hz and 37°C. F) Surfactant after 8 hours rotation at 0.43 Hz and 37°C in the presence of 100 μg/ml TiO₂ nanosized particles. Black arrows show large particle aggregates; lbl - lamellar body like forms; ulv - unilamellar vesicles.