**1. Introduction**

In recent years, the injection of pollutants into the atmosphere as a result of the combustion processes of fossil fuels in industries, power plants, heating systems and vehicles, and the residues of the production processes of nanotechnological industries has undergone a spectacular growth. This opens many questions related to the potential harmful effects of such compounds for human health [1] as is evidenced from the recent statistics of the World Health Organization (WHO) [2], which include long-term exposure to air pollutants as one of the most important sources of cardiovascular diseases, and mortality (around one third of the deaths by strokes, lung cancer, or cardiac diseases are associated with air pollution) [3,4]. Therefore, air pollution should currently be included as a very important public health issue in modern s·ociety [5,6], making it necessary to evaluate the potential impact of pollutants in normal physiological functions, paying special attention to the role of inhaled pollutants [7–9].

Pollutant inhalation is associated with the emergence of pathogenic states and respiratory diseases, e.g., acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary diseases, fibrosis, or lung cancer [10,11]. Therefore, it is critical to perform a careful examination of the effect of particles in respiratory function [12].

**Citation:** Guzmán, E. Fluid Films as Models for Understanding the Impact of Inhaled Particles in Lung Surfactant Layers. *Coatings* **2022**, *12*, 277. https://doi.org/10.3390/ coatings12020277

Academic Editor: George Kokkoris

Received: 27 December 2021 Accepted: 16 February 2022 Published: 19 February 2022

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**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Among pollutants, fine particulate matter (PM2.5, particles with diameter ≤2.5 μm) is counted as one of the most important sources of respiratory diseases [13–15]. This type of particulate matter can be inhaled and transported along the respiratory tract to the alveoli, where they interact with the lung surfactant (LS) film [16,17], modifying the LS chemical composition and lateral organization upon incorporation, which in turn alter LS functionality. Furthermore, the interaction between LS and particles can induce different pathways which favor particle translocation towards other tissues and organs [18,19]. This may be the origin of several adverse health effects [20,21].

LS is a mixture containing mainly lipids and proteins which overlays the inner wall of alveoli in mammals (i.e., the alveolar lining) [7,22], contributing to two very important physiological aspects: (i) regulation of the mechanical properties of the alveoli during breathing, preventing alveolar collapse during exhalation [23–25], and (ii) protection against inhaled matter, mainly pathogens (e.g., SARS-CoV-2) [7,26–31]. Therefore, any dysfunction on the LS mechanical properties or modification of its composition may be harmful for health [32–38]. However, to date there is an important lack of knowledge about the true impact of inhaled particles in the respiratory cycle, even though it is clear that the deposition of particles on LS alters the breathing mechanics, the use of particles, even inhaled ones, in therapy and diagnosis is undergoing continuous growth [39–43]. Therefore, it is necessary to deepen the current understanding of the interactions of different types of particulate pollutants with LS for predicting their potential toxicity [44–46]. However, it is not always easy to perform a direct evaluation of the interaction of pollutants with LS under physiological relevant conditions, and most in vivo tests are invasive, including exposure to particles followed by bronchoalveolar lavage or even the sacrifice of small animals [15,47,48]. This makes it necessary to seek non-invasive in vitro approaches providing a first evaluation of the impact of inhaled particles in the physico-chemical properties of LS films [49–52].

The use of models based on fluid films of different lipids or mixtures of lipids and proteins at water/vapor interfaces, or their deposits onto solid surfaces as Langmuir– Blodgett or Langmuir–Schaefer films, is widespread for the evaluation of the impact of specific chemicals on the activity of different biological systems under physiological relevant conditions [49,53–64]. Therefore, the use of interfacial science may help in the evaluation of the effect of inhaled pollutants or particles in LS films [58,59,65–68] by exploiting the importance of different physical and chemical aspects of the liquid/vapor interface formed between the alveolar lining and the gas contained in the alveolar cavity in LS physiology [69,70]. It should be stressed that studies using model systems are in most of the cases an oversimplification, allowing mimicking of only some specific physicochemical aspects related to the complex biophysical function of LS film. Furthermore, the most common practice relies on the use of models including a limited number of components, i.e., minimal systems, which many times makes it difficult to extrapolate the results obtained by using model systems to the real in vivo situation. On the other side, model systems do not include, in most of the cases, some specific hydrodynamics and aerodynamics aspects guiding the interaction between LS and inhaled particles under true in vivo conditions.

It should be noted that the limitations associated with the use of model systems based on fluid interfaces for evaluating the potential toxicity of inhaled particles upon their interaction with LS cannot hide the many possibilities offered by these types of model systems for preliminary assessment of the harmful effects of inhaled pollutants in the normal respiratory function [59,65,67]. This review tries to provide an updated physicochemical perspective about the current understanding on the potential toxicity of inhaled materials upon interaction with LS films, which requires the introduction of some general points related to the LS performance, including its chemical composition and the role of such composition in LS interfacial properties. Furthermore, considering that the aim of this work is focused on the incorporation of inhaled particles into LS films, some general aspects of such process will be also discussed. On the other side, considering that the review tries to provide insights into the utility of fluid films as models for studying the impact of particles

on LS performance, it is necessary to introduce the most common experimental tools used for exploring the interaction of LS with particulate matter, and the chemical composition of such models together with the influence of the physico-chemical characteristics of the particles in the model properties. The last part of the review will highlight some relevant information about the interaction of particles with LS extracted from the exploitation of the tools offered for the interfacial science.

#### **2. Lung Surfactant from Chemistry to a Functional Layer**

LS is a very complex mixture containing lipids (around 90% of the total weight) and different proteins (comprising about the 10% of the total weight) [24,71], with the specific proteolipidic composition of LS playing a key role and being essential for the control of its structure, properties, and function. This means that the specific composition of LS determines the formation of a surfactant film at the surface of the liquid film covering the alveolar epithelium, contributing to the surface tension reduction and facilitating the respiratory mechanics [32,72]. Furthermore, the formation of the LS film acts as the main biological barrier protecting against the entrance of inhaled particles into the mammal bloodstream. Figure 1 presents a scheme summarizing the average composition of mammal LS.

The lipid fraction includes three major components, phospholipids, cholesterol, and neutral lipids, corresponding to around 79%, 8%, and 5% of the total weight of LS, respectively. Among phospholipids, zwitterionic phosphatidylcholines (PC) emerge as the most important contribution to the average composition of LS (in the range of 60–70% of the total weight of LS), with anionic species, such phosphatidylglycerols (PG) and phosphatidylinositols (PI) together with other minor components, e.g., phosphatidylethanolamines (PE) and sphingomyelin, appearing in smaller concentrations [73,74]. A more detailed analysis of the average phospholipid composition of mammal LS shows that 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) is the main component of the lipid fraction, and in turn, of the LS (in the range 40–50% of the total LS weight) [75]. It should be noted that the exact content of DPPC in LS is inversely proportional to the rate of surface area change, i.e., the respiratory rate, occurring during breathing [71]. Other phosphatidylcholines that appear commonly in mammal LS are 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-palmitoleyl-sn-glycero-3-phosphatidylcholine, and 1-palmitoyl-2-myristoilsn-glycero-3-phosphatidylcholine. It should be noted that the specific proportion between DPPC and other phosphatidylcholines is correlated to the specific respiratory rate and the alveolar development [73,76–79]. Beyond phosphatidylcholines, anionic phospholipids, e.g., phosphatidylglycerols and some phosphatidylinositols, account for 10% of the total

weight of the LS, with an absence of myristoyl components and a low concentration of dipalmitoyl among these types of compounds [73,80]. The presence of anionic PG and PI presents a very important role in the interactions between the lipid fraction and the cationic surfactant proteins (SP-B and SP-C) [22].

The presence of cholesterol is also a very important feature in LS, allowing the modulation of lipid packing in LS structures, and hence any change of the cholesterol concentration may dramatically impact the lateral organization and functionality of LS [81]. Furthermore, the presence of neutral lipids in LS, e.g., cholesterol esters, triglycerides, diglycerides, and free fatty acids, is very important for surfactant properties, and its content has been modulated during evolution for optimizing the activity of the surfactant [82].

The protein fraction is commonly formed by four proteins, the so-called surfactant proteins (SP), SP-A, SP-B, SP-C, and SP-D, which are named following the order of their first detection. Furthermore, some additional proteins, e.g., SP-G and SP-H, can appear in specific mammal species [83]. Among the four most common proteins, there are two oligomeric fibrous ones with hydrophilic characters (SP-A and SP-D), presenting a very important role in the innate defense of the lung. Furthermore, they perform a certain role in maintaining the alveolar surfactant homeostasis [84]. On the other side, SP-B and SP-C are hydrophobic proteins, playing a very important role in the control of the interfacial properties of LS, and hence in its functionality [75,85].

SP-B is a positively charged polypeptide with a molecular weight of about 8.7 kDa, adopting α-helical structure [86]. This protein performs a very important role on the LS adsorption at the liquid/gas interface, and in the dynamics exchange between the interfacial LS layer and the subphase enriched in compounds with low surface activity [86,87]. Furthermore, SP-B plays a very important role in the storage of LS modulating the assembly of lipids into the lamellar bodies, and their deficiencies are lethal [88]. On the other side, SP-C is a small hydrophobic protein (molecular weight around 3.7 kDa) with helical structure, and their deficiency is not lethal but can induce chronic respiratory disorders [72,89]. SP-C is associated with the promotion of vesicle fragmentation helping in the membrane curvature [90,91].
