**7. Some Experimental Results of the Interaction of Particles with Interfacial Lung Surfactant Models**

The understanding of the potential harmful effects associated with the incorporation of particles in LS layers requires a deeper analysis of the impact of particles in the tensiometric properties and dynamic response of the interfacial films, as well as their effect on the lateral organization and structure. Furthermore, it is also very important to evaluate the distribution of particles between phases with different orders because it can impact on the translocation of particles across the pulmonary fluid and the clearance processes. This section will review the effect of different types of particles on the behavior of LS models, paying attention mainly to realistic systems including lipids and the surface-active proteins.

The incorporation of particles, mainly those of hydrophobic nature, into LS films is commonly associated with a partial inhibition of the LS function. This was evidenced in the study by Valle et al. [227] where the interaction of polymer particles with LS films was evaluated. These particles lead to a contraction of the area available for LS molecules, i.e., shift the isotherm to more compressed states, which significantly alter the ability for surface tension reduction (see Figure 10a). The modification of the tensiometric properties as a result of the incorporation of particles is also reflected in changes in the lateral organization of the molecules at the interface as evidenced by AFM micrographs of Langmuir-Blodgett films at different surface pressures (see Figure 10b). These images evidence that particle incorporation makes the lateral packing of the molecules difficult, hindering the nucleation and growth of domains containing ordered phases, which disturbs the monolayer-tomultilayer transition. On the other side, the analysis of the AFM images also evidences a higher tendency of particles to aggregate into LS layers as their hydrophobicity increases, which may be a signature of the important role of hydrophobicity in the control of the particle retention and translocation across the pulmonary fluid. Last but not least, the

incorporation of particles increases the area of the hysteresis loop of the compressionexpansion cycles of the LS layers, which is expected to present a critical impact on the normal function of LS (see Figure 10c). Recently, Beck-Broichsitter et al. [236] evidenced that the inhibition of the LS activity upon particle deposition is strongly correlated to their ability to sequester the surface-active proteins. Therefore, the shielding of the particles to avoid the formation of an LS corona reduces their harmful effects and allows their exploitation as carriers for inhalable drugs.

**Figure 10.** (**a**) Modification of the compression isotherms of an LS model for the compression isotherm of an LS model. (**b**) AFM micrograph showing the lateral structure of Langmuir–Blodgett films at three different surface pressures of the LS model and LS models upon the incorporation of three different types of hydrophobic polymer particles. All 2D images are shown at a resolution of 50 μm × 50 μm and have a z-range of 5 nm. The high-pressure images are shown in 3D to capture the topographic contrast between the particles and multilayer structures. The presence of NPs, denoted by white arrows, increases with increasing hydrophobicity. (**c**) Compression–expansion cycles for the pure LS model and LS model upon the incorporation of three different types of hydrophobic polymer particles. Dynamic cycling was conducted under physiological temperature (37 ◦C) and cycling speed (3 s/cycle). Reprinted from Valle et al. [227], with permission from American Chemical Society, Copyright 2014.

The deposition of hydrophilic hydroxyapatite particles leads to similar effects than those discussed above for hydrophobic polymer particles [69]. In addition, there is a strong time dependent behavior in the inhibitory character of the LS function as a result of the inhalation of hydroxyapatite particles, reaching the maximum inhibition after 7 h of exposure. It should be noted that even though hydroxyapatite particles are effectively cleared from LS films, they modify the lateral packing of the LS molecules at the fluid interface by hindering the formation of condensed phase, altering the remodeling process of the film and the reservoir formation. LS films at physiologically relevant conditions present a morphology characterized by the presence of a homogeneously distributed fluid multilayer in which insertions of DPPC domains appear. This structure changes upon the deposition of particles on the LS films, with the appearance of crystalline folds along the direction of lateral compression, which leads to the inhibition of the LS performance.

It should be noted that the interaction of particles with LS models containing proteins emerges very different in most of the cases to what happens for model mixtures in absence of surface-active proteins. The role of these proteins in the formation of the LS corona on the surface of the particles is essential for the inhibitory role of particles [236]. The important role of the proteins is clear considering that in most cases the interaction of hydrophilic particles, e.g., silicon dioxide, Fe3O4, or titanium dioxide, with models based only on lipids, takes the surface pressure-area per molecule to more expanded states. This shifting is enhanced with the increase of the particle concentration, which may be explained considering that the interaction occurs through electrostatic particles, with the particles being retained as granular domains within the monolayer [117,119,188,223].

The importance of the model composition is also supported by the results obtained by Tatur and Badia [115] using a multi-technique approach (surface tension measurements, ellipsometry, Brewster angle microscopy, and AFM of Langmuir-Schaeffer films). They compared the effect of alkylated gold particles on the interfacial behavior of DPPC and discovered that the particles do not alter the tensiometric properties of Survanta films, but they lead to a strong modification to those of the DPPC layers, changing both the phase behavior and the lateral packing of the molecules. Thus, gold nanoparticles hinder the nucleation and growth of condensed phase domains, leading to the change of the shape of the domains from a multilobe geometry for pristine DPPC monolayers to a circular one in the presence of particles. This is similar to what was reported for the incorporation of silico dioxide particles into DPPC films [179]. The different effect of gold particles on DPPC and Survanta films can be understood by considering that their accumulation occurs within the disorder region, and this leads to the incorporation of particles within the phase containing the surface-active proteins. This minimizes their effect on the condensation of ordered phases when Survanta films are considered. The above results suggest that alkylated gold particles present a rather limited inhibition of the LS function under the experimental conditions. However, a true biophysical interpretation of the results should consider its effect under conditions mimicking the physiological one. This becomes very important because Hossain et al. [237] reported the inhibition of LS function as a result of the interaction with gold nanoparticles. These make the reduction of the surface tension of the water/vapor interface difficult, reducing the lateral packing of LS molecules at the interface and dragging LS molecules into the adjacent subphase

Tobacco smoke results in similar inhibition of the LS performance as particles, as was evidenced for Survanta and Curosurf films under physiological relevant conditions [238]. Thus, the response of LS layers after exposure to tobacco smoke to consecutive compressionexpansion cycle evidences a clear reduction of the efficiency of the compositional remodeling process, which agrees with the reduced formation of reservoir evidenced from the analysis of the Langmuir–Blodgett deposits. Furthermore, the tobacco smoke reduces the ability of the LS to reduce the surface tension under compression conditions, increasing the respiratory work. The comparison of the effect of tobacco smoke with that associated with the vapor ejected by electronic cigarettes evidenced unexpected results [172]. On one side, both alter the lateral packing of LS film, whereas on the other side only tobacco smoke modifies the tensiometric properties of LS films, resulting in a premature collapse of the layer. These differences can be understood only by considering the different chemical nature of the vapor obtained from each type of cigarette. The influence of tobacco smoke can be correlated to the effect of other types of hydrophobic carbonaceous particles (nanotubes or nanohorns), which alter the viscoelastic response of LS layers (Survanta) under dynamic conditions, enhancing the monolayer rigidity. However, the effect of hydrophilic carbonaceous particles is rather limited which may be explained considering their effective clearance [161].

The inhibition of the LS function is not only associated with particle inhalation, with the exposure to other chemicals (trimethoxyoctylsilane, methyl 3-oxo-2-pentylcyclopentaneacetate, and diisopentyl ether) also being associated with the emergence of acute inhalation toxicity as was evidenced by combining interfacial science techniques and biophysical methodologies. It should be noted that the understanding of the potential inhalation toxicity associated with these types of chemicals is very important because LS is the main target of impregnation spray products [239]. The interaction of LS layers with the above products leads to a partial inactivation of the LS function, resulting in a fluidization of the condensed phases due to the insertion of the chemical between LS molecules which weakens the cohesion in the layer and reduces the stability of the LS function. There are many other chemicals, including benzalkonium chloride and cetylpyridinium chloride, that present a strong inhibitory characteristic of the LS function. These molecules alter the lateral organization of the molecules at the water/vapor interface, affecting the ability of LS to reduce the surface tension [240].
