**9. What Can We Learn Using Model Lung Surfactant Films?**

The effect of particle incorporation into LS films is the emergence of specific dependency on the specific nature of the considered particles, and the use LS model. However, most of the studies present some general features that are relevant for extracting biophysical relevant information. In particular, the incorporation of particles into LS films worsens the mechanical properties of the surfactant-laden interface by hindering the reorientation and lateral packing of the surfactant molecules at the water/vapor interface. This alters the compositional remodeling of the interfacial layer. Furthermore, the use of model systems evidences that the interaction of LS with particles results many times in the formation of a

surfactant corona on the particles surface, which leads to a compositional modification of the LS, and can facilitate the translocation of the particles beyond the alveoli, resulting in their transport along the bloodstream to different organs and tissues. Thus, the interaction of particles with LS can alter and inhibit the LS functionality through different mechanisms.

Moreover, the formation of the surfactant corona can also modify the dynamic of material exchange between the interfacial layer and the adjacent fluid subphase, which alters the particle clearance during breathing. However, it is necessary to obtain a further understanding on how specific components of LS may influence the biological alterations induced by particles, and in particular the influence of the adsorption of surfactant proteins on the particle fate, and the physiological function of LS.

The use of LS models allows inferring that the inhalation, and subsequent deposition of particles on LS leads to an important alteration of the normal physiological respiratory function. However, it is necessary to deepen knowledge of the specific effects induced by particles with different properties (charge, wettability, chemical nature, morphology, or size) for obtaining a suitable evaluation of the potential harmful effects associated with inhaled pollutants. This is a very important for unraveling the possible elimination pathways of the particles after their deposition on the LS film. Furthermore, the understanding of the specific effects associated with particle nature is very important in the modulation of the in vivo interaction of LS and pollutants.

In summary, the use of fluid films as LS models presents an important role in the safety evaluation of particulate pollutants. However, there are many open questions that require refining of some specific physico-chemical and mechanical aspects of the currently available models.

### **10. Conclusions**

This review analyzes some of the most relevant colloidal and interfacial approaches exploited for the in vitro evaluation of the potential toxicity of inhaled pollutants. It is true that the results obtained by using these types of tools are often times difficult to extrapolate to a true biophysical situation, they are useful for a fundamental understanding of some of the physico-chemical aspects associated with the harmful effects of particulate matter in the LS function. In particular, the use of model based on monolayers at fluid interfaces can help to understand how the deposition of particle leads to either inhibition, inactivation, or both, of the LS function due to the alteration of the packing/organization of the molecules at the interface. This hinders the formation of ordered phases, and inhibits the compositional remodeling of LS layers at low values of the surface tensions, which reduces the LS ability for reaching surface tension values close to zero, increasing the mechanical work associated with breathing. However, the use of colloidal and interfacial models makes it difficult to mimic the entire respiratory cycle and its modification by particles, thus they provide very limited access to aerodynamic and hydrodynamic aspects of the problems which many times are very important aspects for understanding the true effect of inhaled materials in respiratory physiology. Despite the clear limitations associated with the use of model systems, it is clear that these types of systems provide a general perspective of the potential risks and hazards associated with particle inhalation. However, a complete picture of these aspects can only be obtained by combining physico-chemical models with complementary biophysical and medical studies.

**Funding:** This work was funded by MICINN (Spain) under grant PID2019-106557GB-C21 and by the E.U. in the framework of the European Innovative Training Network-Marie Sklodowska-Curie Action NanoPaInt (grant agreement 955612).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
