**4. Surface Science Approaches for Evaluating the Interaction of Particles with the Lung Surfactant Film: Tools and Models**

## *4.1. Experimental Tools*

The use of colloidal and interfacial approaches allows overcoming some of the main limitations associated with the in vivo evaluation of the interaction between inhaled particles and LS, providing important insights on the most fundamental bases governing the interaction of particles with LS, avoiding the use of invasive methodologies [58,59,66].

The use of Langmuir film balances for studying spread films of lipids or lipid-protein mixtures at liquid/vapor interfaces are probably the most exploited approaches for deepening on the knowledge of LS mechanics, and its modification as results of the incorporation of particles or different types of chemicals, providing very insightful information on the quasi-static mechanical properties of LS films [58,144]. It is true that the studies based on the use of Langmuir film balances do not provide true biophysical information. However, they help in the understanding of several aspects with relevance for the incorporation of particles into LS films in terms of the modification of the surface pressure of pristine LS layers as a result of the incorporation of particles, i.e., the changes in the surface pressure vs. molecular area plot for the interfacial film, the so-called interfacial isotherm [108,117,118,128,129,145].

The use of Langmuir film balances also allows the study of the rheological response of interfacial layers under compression-expansion cycles (oscillatory barrier experiments) which are somehow reminiscent of the deformations of the alveoli during breathing. During these experiments, the surface pressure is monitored by using a surface balance fitted with a contact probe, normally a Wilhelmy plate, whereas the area available for the film is modified by the coupled motion of two barriers arranged parallel in opposite extremes of the trough [146,147]. These experiments require surface pressure changes occurring in comparable time-scales to the area changes. Despite the many possibilities offered for the use of Langmuir film balance, it should be stressed that the probed frequencies and deformation amplitudes are far from that what occur during normal breathing, and in turn they only give semi-quantitative information that can be exploited for evaluating the incorporation of particles into LS films.

Langmuir film balances can also be exploited for transferring the interfacial film from the surface of the liquid to a solid substrate by the Langmuir–Blodgett or Langmuir– Schaefer methods, which makes an ex situ study possible of the interaction of the LS film with particles using microscopy techniques, commonly atomic force microscopy (AFM). These types of studies provide insights on how particles modify the interfacial morphology

of LS films [148–150]. Figure 4 shows sketches of the Langmuir film balance and the two alternatives for depositing films on solid surfaces.

**Figure 4.** (**A**) Scheme of a Langmuir film balance fitted with a contact probe (Wilhelmy plate) and two symmetric barriers. The numbers indicate: 1. Fluid interface, 2. Symmetric barriers and 3. Force balance fitted with a Wilhelmy plate. (**B**) Scheme of the vertical transference of a film from a fluid interface to a solid substrate following the Langmuir–Blodgett method. (**C**) Scheme of the horizontal transference of a film from a fluid interface to a solid substrate following the Langmuir–Schaefer method. Reprinted from Wang et al. [67], with permission from Elsevier, Copyright 2020.

The highly dynamic character of the alveoli during breathing requires deepening of the mechanical characterization of LS layers under dilational deformations mimicking the expiration-inspiration cycles [116,151]. This information is difficult to obtain by using Langmuir film balances as was stated above (further details can be found in the recent review by Ravera et al. [68]). However, the use of techniques based on oscillating drops or bubbles, e.g., pulsating bubble surfactometer or captive bubble tensiometer [152–156], help in the evaluation of the potential impact of the particle incorporation in the mechanics of LS films under realistic simulations of the physiological situation. Thus, it is possible to evaluate the remodeling process and stability of LS films upon their exposure to inhaled particles.

The use of the captive bubble tensiometer relies on the creation of an air bubble suspended in a liquid phase commonly containing the LS which adsorbs to the liquid/vapor interface. Once the LS film is ready at the interface, particles are deposited on the interface by injection in its vicinity through the liquid phase. In some cases, the particles can be premixed with the LS before the formation of the bubble, which provides a less realistic representation of the inhalation process. The use of a captive bubble tensiometer makes it possible to evaluate the fast adsorption of the LS film to the water/vapor interface, and the remodeling processes, as well as the surface activity of LS under changes of the interfacial and its stability [157,158]. Another alternative for evaluating the impact of particles in the mechanics of LS films is the use of a pulsating bubble surfactometer. This technique is based on the creation of an air bubble suspended on a capillary tube inside a chamber filled with LS which undergoes compression-expansion cycles by the action of a piston pulsator [152,157].

Conventional oscillating bubbles have also been used for exploring the effect of particles in LS [159,160]. This approach relies on the oscillation of the bubble surface at a frequency close to that corresponding to the breathing rate (around 15 min<sup>−</sup>1), allowing the evaluation of different relevant properties for the breathing cycle, including the minimum surface tension reached under compression and the area of the hysteresis loop associated with the compression-expansion of the area available, and how these parameters are influenced as a result of the incorporation of particles [68]. Oscillating drops in pendant drop tensiometers also allow obtaining information about the mechanical performance of LS layers under sinusoidal changes of the interfacial area [159,161,162]. Thus, the analysis of the dependence of the dilational viscoelasticity of the layer on the deformation frequency helps in understanding how particles modify the kinetics associated with the exchange of material between the interfacial film and the adjacent fluid layer. This becomes very important because any modification of the remodeling kinetics may be either a signature of inhibition, degradation of the LS activity, or in combination [137,163,164]. Therefore, the combination of experimental results and suitable theoretical models may help in the evaluation of the particle impact in the interfacial relaxation [165]. This is in agreement with the work by Kondej and Sosnowski [166], where the changes in surface viscosity and elasticity obtained against dilational deformations of the interface are proposed as a signature of the inhibition of the LS activity due to the particle incorporation. As an alternative, the analysis of the evolution of the linearity of the rheological response in terms of the total harmonics distortion (THD) is also a very promising tool for evaluating the inhibition of the LS film mechanics [165].

The use of constrained drop surfactometer (see Figure 5) has recently emerged as a very promising tool for studying LS films under conditions which are very close to the true physiological one [70].

**Figure 5.** Sketch of the typical configuration of a constrained drop surfactometer. Reprinted from Valle et al. [70], with permission from ACS, Copyright 2015.

Constrained drop surfactometer consists of a single sessile droplet constrained on a knife-sharp edge pedestal where the LS is accommodated. This configuration avoids surfactant leakage even at the lowest values of surface tension [167], allowing real time evaluation of the volume, surface area, and surface tension of the droplets using a modified axisymmetric drop analysis (closed-loop axisymmetric drop analysis). These act as feedback control systems enabling the direct measurement and modification of the above-mentioned parameters. The main novelty of the use of constrained drop surfactometer in relation to other traditional configurations relies on the possibility to perform precise harmonic oscillatory deformations of the interface with predefined amplitudes and frequencies (up to 0.5 Hz), which is useful for determining the surface dilational modulus before and during the exposure of the LS film to particulate matter [167].

The above discussion shows clearly that the evaluation of the effect of particles on the interfacial properties of LS films is a very interesting tool for deepening knowledge on the effect of pollutants in the dynamic response of the air/liquid interface in the alveoli during breathing [168]. This is possible by evaluating aspects, such as the minimum and maximum surface tension values reached upon compression and expansion, respectively, which provides important insights on the surfactant functionality. However, in recent years, many researches have turned their interests towards the understanding of the relationship existing between the changes in the interfacial packing of LS layers and the worsening of their mechanical properties [22,169]. This is important because as was stated above, the LS present a very complex organization at the interface and in the adjacent liquid phase [170], and the use of techniques such as Brewster angle microscopy (BAM), AFM, surface force apparatus (SFA), ellipsometry, infrared reflection absorption spectroscopy (IRRAS), or epifluorescence microscopy, and their combinations with tensiometric techniques (mainly Langmuir film balances) are very powerful tools for deepening knowledge on the impact of particles on the organization of LS films in the micrometric and submicrometric scale [171–174]. Furthermore, there are more sophisticated tools, including neutron reflectivity or synchrotron grazing angle X-ray diffractions which may also be useful in the evaluation of the impact of particulate matter on LS interfacial organization [108,134,175–182].
