The present data show that nanosized particles, but not microsized particles, induce a dysfunction of pulmonary surfactant. Nanosized titanium dioxide as well as nanosized polystyrene particles at high concentrations can induce a slight pulmonary surfactant dysfunction in vitro. Interestingly, surface area cycling in vitro aggravated the surfactant dysfunction induced by nanoparticles, both by TiO2 NSP and by polystyrene NSP. In addition, biophysical alterations of pulmonary surfactant by TiO2 NSP were accompanied by changes of the surfactant ultrastructure indicating increased surfactant subtype conversion.
A direct interaction between particles and the surfactant constituents is the most likely explanation for the observed surfactant dysfunction. It is well known that phospholipids bind to particles [10, 14, 23] and to TiO2 structures [24, 25]. In this respect, surface area seems to be the major determinant of the observed biophysical and ultrastructural changes. Accordingly, particles with the highest surface area - TiO2 NSP and also reference polystyrene NSP - induced the most prominent alterations. Microsized particles with a relatively low surface area did not induce a surfactant dysfunction in our study.
In separate experiments, we compared equal surface areas by testing very high microparticle mass concentrations. With concentrations of ~10 mg/ml TiO2 MSP and quartz MSP, we observed a strong surfactant dysfunction. However, the experimental conditions were limited because microsized particles at this very high concentration aggregated and rapidly sedimented to the bottom of the test capillary. By this segregation, the phospholipid concentration was not stable which limits the comparison of NSP and MSP at similar surface areas.
Bakshi and coworkers demonstrated a potent pulmonary surfactant dysfunction at low concentrations of ~2 μg/ml gold nanoparticles . In contrast, much higher concentrations of TiO2 NSP were required to induce an increase of surface tension in our experiments. In addition, the degree of surfactant dysfunction was less with TiO2 NSP in our study compared to the gold nanoparticles used by Bakshi et al. Differences in 1) the measuring system, 2) the surfactant preparation and concentration, or 3) the nanoparticles themselves might account for the discrepancy. Both, the pulsating bubble surfactometer (PBS) and the captive bubble surfactometer (CPS) are able to evaluate low surface tensions  while the CPS is regarded to yield even lower surface tensions  which makes differences in the device an unlikely explanation. Regarding surfactant preparation and concentration, we used Curosurf®, a natural surfactant derived from minced porcine lungs  while a semisynthetic surfactant composed of two phospholipids plus SP-B was used by Bakshi. It is unlikely that differences in the surfactants are solely responsible for the different effects seen with gold nanoparticles and TiO2 NSP. Both surfactants have been demonstrated to have excellent surface activity and to achieve very low surface tensions under compression at the concentrations used. The most likely explanation for the potent dysfunction in the study by Bakshi seems related to the material properties (size/surface) of the gold nanoparticles. Since the gold NSP had citrate groups on their surface, aggregation is mostly avoided . In contrast, pure TiO2 nanoparticles highly aggregate. Although the surface area is not known for the gold NSP from Bakshis study, it is likely that the surface area per mass unit is higher for the citrate coated gold NSP than for the TiO2 NSP. This could explain the more potent induction of surfactant dysfunction by gold NSP compared to TiO2 NSP because surfactant components could be bound to the large gold nanoparticle surface area making them unavailable for lowering surface tension at the air-liquid interface.
In an attempt of direct comparison between TiO2 NSP and the gold nanoparticles by Bakshi (~15 nm), we tested commercially available gold NSP with citrate coating (5 nm) in single experiments. Interestingly, at equal mass the surfactant dysfunction by gold NSP was stronger compared with TiO2 NSP. However, the dysfunction was less compared with data from Bakshi et al., but this discrepancy can be accounted to differences in surface area of the gold NSP or the surfactant preparations used in both studies.
The in vivo conversion of surface active LA to inferior SA can be simulated in vitro by surface area cycling . By this technique, the impact of meconium, serum proteins, or surfactant proteins during the surfactant conversion process have been studied [19, 30–32]. We assessed the effect of TiO2 NSP on the conversion process. Importantly, a dose-dependent increase in surface tension was obtained. Remarkably, this effect was much stronger than the direct biophysical effect of TiO2 NSP without cycling. TEM pictures demonstrated that the occurrence of unilamellar vesicles was independent from NSP presence. Possibly, binding of NSP to SP-B and subsequent loss of SP-B from the air-liquid-interface could explain the loss of surface activity following surface area cycling in the presence of NSP. In vivo, SP-B becomes cleaved by a serine active carboxylesterase called convertase [33–35]. However, Curosurf® is prepared by chlorofom extraction and hence does not contain convertase . Therefore, intact SP-B should be present in Curosurf® following surface area cycling. We speculate that free SP-B could interact with TiO2 NSP which in turn becomes depleted leading to disturbed surfactant function. High ability of SP-B to bind to surfaces during surface area cycling was shown before when binding of SP-B to tube walls was investigated during surface area cycling . Unfortunately, we were not able to provide direct evidence of binding of SP-B to TiO2 NSP by TEM due to methodological limitations.
Admittedly, inhaled particles act directly on the surfactant layer at the air-liquid interface and not primarily through the subphase as in our in vitro experiments. However, after deposition at the air-liquid interface the particles subsequently become dissolved in the epithelial lining layer and interfere with the dynamic process of phospholipid arrangement at the interface. Therefore, the assay system with the pulsating bubble surfactometer is at least capable to demonstrate differential effects of nanoparticles versus microparticles in phospholipid suspensions when particles interfere with the formation of the surfactant layer from the hypophase. It is very well conceivable that the initial effect of particles might even be greater when they are reaching the interface directly.
Although we have demonstrated that TiO2 NSP elicited biophysical and structural changes of surfactant in vitro, the in vivo relevance has to be scrutinized because the particle concentrations that we found effective in vitro can hardly occur in vivo. With the human alveolar surface area of ~100 m2 and assuming an average thickness of the alveolar lining fluid of approximately 200 nm , the amount of alveolar lining fluid can be assessed as ~20 ml. In accordance, the epithelial lining fluid has been suggested to be 6 ml/L total lung capacity, resulting in 40 ml in man . With the assumption of a particle concentration of 100 μg/m3, which can occur in polluted inner cities, and an alveolar deposition rate as high as 50%, the amount of particles deposited per day would be ~360 μg. At steady state, this would result in a concentration of ~10 μg nanoparticles per ml alveolar lining fluid. This particle concentration is far below what has been demonstrated to cause a surfactant dysfunction in our study. In addition, clearance of particles and secretion of newly synthesized surfactant would further improve this particle/surfactant ratio and consequently question whether nanoparticles can cause a surfactant alteration under these conditions in vivo. This view is supported by our experimental evidence in rats. Following inhalation of TiO2 particles that were aerosolized and adjusted to result in the highest technically possible alveolar deposition of 60 μg particles per animal, surfactant ultrastructure was found unaffected in vivo. Assuming an alveolar lining fluid in rats of approximately 70 μL , the in vivo particle concentration in the epithelial lining fluid would have been approximately 53.5 μg/mL (normalized to 1.5 mg/ml phospholipids and assuming static conditions). Noteworthy, the local concentration at the air-liquid interface was probably much higher suggesting that no changes of surfactant ultrastructure occur in vivo under acute maximal TiO2 particle exposure.
Although these considerations suggest that the impact of TiO2 NSP on surfactant function in the human lung is highly unlikely to cause adverse effects in healthy individuals, in diseased subjects, however, additive effects of NSP on pulmonary surfactant function and ultrastructure have to be taken into account. For example, it has been demonstrated that a pulmonary surfactant dysfunction can be found in various airway diseases like asthma , cystic fibrosis , or after lung transplantation . In particular, leakage of plasma proteins into the airway lumen is known to induce a surfactant dysfunction [41, 42]. Importantly, NSP can induce [43–45] or enhance [46, 47] pulmonary inflammation which is accompanied by protein leakage. This in turn could lead to a surfactant dysfunction in vivo. Moreover, NSP are able to induce oxidative stress and lipid peroxidation [48–50]. Oxidative stress with lipid peroxidation can induce an increase in surface tension [51, 52]. In addition, NSPs emitted by engines are contaminated with alkanes and sulfates  and it is known, that eicosane, a specific n-alkane constituent of diesel exhaust NSPs, can affect the biophysical surfactant function . Therefore, nanoparticles might amplify alterations of the pulmonary surfactant system, particularly under predisposed conditions of airway inflammation.