**5. Evaluating the Interaction of Lung Surfactant and Particles: Methodological Approaches**

The investigation of the interaction of particles with LS films requires, in most cases, to put into contact a film preformed at the water/vapor interface and the particles, which can be commonly done by aerosolization or deposition. In particular cases, the studies are performed by premixing the lung surfactant and the particles before the preparation of the interfacial films [32]. The use of these different approaches can decisively impact the biophysical interpretation of the experimental results [206]. Furthermore, the existence of different approaches for incorporating particles into LS layers makes it difficult to compare the different impacts found for similar particles in different studies. Thus, the incorporation of hydrophilic particles into LS layers is commonly explored by spreading the LS model film onto a subphase containing a dispersion of particles [119,188], or by the injection of the particles into the subphase once the LS layer is preformed at the water/vapor interface [116,207]. These methodologies are far from the real situation that is found during particle inhalation. However, they emerge as very useful alternatives for evaluating the direct interaction of particles with LS layers [67].

The situation becomes even more complex when the interaction of hydrophobic particles with LS layers is considered. In this case, there are multiple possibilities for incorporating particles into LS films. The first approach used for the incorporation of hydrophobic into LS films relies on the co-spreading at the pristine water/vapor interface of a mixed dispersion containing the LS model and the particles [42,208]. Thus, it is possible to tune, at will, the exposure dose in relation to the LS concentration, ensuring that the exposure of the surfactant to the particles is high enough. However, the obtained films are probably very far from what are expected once inhaled particles interact with LS layer, the formation of particles decorated with a surfactant corona emerging even before the formation of the LS layers at the water/vapor interface. A more realistic approach considers the deposition of particles at the interface from the airside once the surface layer is formed at the water/vapor interface [118,133], which leads to a more realistic picture of what happens in the LS film upon pollutant inhalation. However, organic solvent is used for the spreading of either the LS layer, the particles, or both [206,209], which may modify the LSparticle interactions, or the lateral packing and homogeneity of the film, and the difficulties associated with the mimicking of the specific mass transport conditions occurring during in vivo breathing are two main drawbacks of the studies using the approach discussed above [67,70,130,131].

Aerosolization of the particles onto a preformed LS film by using a dry power insufflator can provide a more realistic representation of the inhalation process, even though the use of organic solvents continues being necessary for spreading the LS layer [199]. Figure 6 presents sketches of three commonly used approaches for the study of particles with LS models, as well as examples of the differences in the surface pressure-area per molecule isotherms obtained using different preparation procedures.

**Figure 6.** Sketches representing the three different approaches used for incorporating particles into LS models layers based on DPPC, and the differences in the surface pressure-area per molecule isotherm obtained depending on the procedure used for preparing the mixed layers. Adapted from Miguel-Diez et al. [206], with permission from Elsevier, Copyright 2019.

The above discussion considers the study of the interaction of particles with spread monolayers of LS models obtained by mixing different raw materials in an organic solvent. However, a very different situation emerges when commercial LS formulations are used as models. These are commonly supplied as saline aqueous dispersions of LS components as vesicles and micelles of lipids associated with LS proteins. Therefore, the use of these types of formulations require the preparation of interfacial films by the direct adsorption of the LS components from the bulk dispersion to the aqueous solution/vapor interface. This requires depositing the particles on the interfacial film by injection into the aqueous subphase after the formation of the interfacial layer (hydrophilic particles) or by spreading through the airside onto the formed layer (hydrophobic particles). On the other side, hydrophilic particles can also be premixed with the LS dispersion before forming the interfacial film. The latter leads to the co-adsorption of LS molecules and particles at the liquid/vapor interface [210]. It should be stressed that commercial LS formulation can also be extracted in an organic solvent, and then applied by spreading at a pristine/water vapor interface [211].

It is true that most of the studies using LS models for exploring the impact of particles in the respiratory mechanics present a situation that appears very far from what happens when the pollutants are inhaled. However, these studies are very useful for gaining an important understanding of the most fundamental physico-chemical bases of the interaction of inhaled pollutants and LS films, and their potential harmful effects [59,65,66].
