*4.2. Chemistry of Lung Surfactant Models*

The methodologies used for evaluating the interaction between pollutants and LS components rely on the use of different models, ranging from simple lipid monolayers to complex surfactant extracts obtained from bronchoalveolar lavage fluid. Therefore, it is necessary to know the main differences of the physico-chemical properties of the chosen model with respect to the true LS to draw conclusions with biophysical relevance. Therefore, any LS model should mimic some of the physical properties related to the physiological function of LS: (i) ability to decrease the surface tension of the water/vapor interface down to a quasi-null value (<2 mN/m) upon reduction of the interfacial area, (ii) effective compositional remodeling during compression at very low surface tension values, and (iii) fast re-adsorption and respreading during expansion of the area of the interface [7,58,66,67].

DPPC fulfills the first requirement, which has pushed the use of DPPC monolayers as minimal models for understanding some physico-chemical aspects related to the impact of particles in LS films. Furthermore, the interfacial behavior of this lipid has been extensively studied by many researchers [59,66,175,183–187]. However, the use of DPPC as a model for understanding the performance of LS is rather limited because its inefficiency in the reservoir formation and its slow respreading at the interface during expansion. This has driven the research on the use of more complex models, including DPPC in combination with other lipids or fatty acids, e.g., palmitic acid, cholesterol or DOPC (1,2-dioleoylsn-glycero-3-phosphocholine), among others [117,136,165,173,188–191], which provide a simplified picture of the complex behavior of LS under physiological conditions. The use of these models can provide a deeper understanding of the behavior of LS films because some studies have evidenced the important role of specific lipids, e.g., 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1 glycerol)) (POPG), on the modulation of the phase behavior of LS film [173,189,190,192]. On the other side, it has been reported that the inclusion of DOPC in LS models provides a suitable environment for gaining information of the remodeling process, contributing to the squeezing-out of lipids and reservoir formation upon compression, and to the respreading of the LS upon expansion [193].

It should be noted that the biophysical interpretation of the results obtained from studies using LS models composed by single lipids or their mixtures is very difficult, especially because the absence of the surface-active proteins makes it difficult to control the exchange of molecules between the liquid/vapor interface and the adjacent aqueous subphase during the compression-expansion cycles of the interfacial layers, and hence the control over the formation of reservoirs and the re-adsorption/respreading of material at the fluid interface becomes very poor, which are very critical processes for the normal physiological function of the LS related to the surface properties and mechanical stability of the interfacial layer [22,104,106,194]. Therefore, the use of most realistic models, based on natural extracts or laboratory mixtures, having a composition similar to that what is found in mammal LS is required for a biophysical quantification of the potential effects of particles in LS films. This has been solved by using different commercial LS formulations (clinical LS) as models [195]. These types of surfactants are commonly used in surfactant replacement therapy (SRT), and for treating neonatal respiratory distress syndrome (NRDS) [196,197], having gained interest as palliative treatment for reducing the impact of SARS-CoV-2 in respiratory physiology [30,198]. Table 1 summarized some of the currently used clinical lung surfactants and their origins.

**Table 1.** Summary of the most extended clinical lung surfactants, together with their type, origin, and producer.


The importance of the use of realistic LS models is easily understood considering the differences on the effect of particles in the behavior of interfacial films of DPPC and commercial LS formulation [199]. In particular, DPPC films containing particles do not easily undergo any remodeling process, emerging as a fast destabilization of the interfacial film when the surface tension reaches a low value. This pushes the monolayer to a premature collapse [133,200], which drives the formation of bilayer folds and other 3D structures. These structures are not easily re-adsorbed into the interfacial film upon expansion. However, the presence of other lipids and the surface-active proteins SP-B and SP-C results in a minimization of the destabilization of the interfacial film. Thus, whereas from the studies of the interaction of particles with DPPC films a clear inhibition of the LS activity may be expected, this can be, at least in part, ruled out from similar studies in which commercial LS formulations are used as model [199,201–203]. Similar conclusions can be extracted from the analysis of the minimum surface tension that can be reached by LS model in presence of particles. Thus, the incorporation of particles into interfacial films of commercial LS formulations leads to a smaller increase of the minimum surface tension reached upon compression than when particles are incorporated in DPPC layers. This can be understood considering that the presence of surface-active proteins favors the remodeling process of the interfacial layer, and in particular the reservoir formation and the re-adsorption/respreading kinetics of the squeezed out material during the expansion of the interfacial area [115,199].

The Tanaka mixture containing only lipids (DPPC, POPG, and palmitic acid in weight ratio 68:22:9) emerges as a very simple and robust model for study involving the interfacial properties of LS [204]. This is possible because the addition of POPG to DPPC monolayers plays a crucial role in the modulation of the interfacial compressibility of the interfacial film by reducing the rigidity of pristine DPPC films, which is essential for the LS packing and its ability to reduce the interfacial tension. Furthermore, the presence of POPG has a very important contribution in the formation and stabilization of reservoirs. On the other side, palmitic acid is essential for the fluidization of films at high values of surface tension, making the molecular rearrangement in the LS films easy. Furthermore, palmitic acid contributes to the interfacial packing, improving the film rigidity at low surface tension values, avoiding a premature collapse.

Despite that many studies dealing with LS models consider the use of single monolayer, the LS complexity can be modelled by using other types of colloidal structures, including bilayers or multilayers [49]. This presents interest for gaining additional insights into the LS behavior and how this is modified by particle incorporation, especially considering the important role of bulk structures, e.g., reservoirs, in the exchange of material between the interface and the adjacent fluid phase during the compression-expansion cycles [205].

From the above discussion, it is clear that there are many possibilities for exploring LS films, especially from a mechanical point of view. However, the used of models with biophysical relevance should consider two main aspects: (i) the temperature used should be close to the physiological one (37 ◦C) and (ii) the specific hydrodynamic conditions of the experiments which govern the Marangoni flows within the interfacial film, and between the interface and the adjacent fluid phase [65,66,107,109,130,131].
