*Lung Surfactant Interfacial Activity: A Key Aspect for the Respiratory Function*

The interfacial activity of LS is closely correlated to the phospholipid ability for self-organizing at the interface but also to the LS composition, temperature, and lateral pressure [72,92]. Thus, below the melting temperature (temperature for the order-disorder transition), the lateral mobility of phospholipids is hindered due to the strong interfacial packing, whereas once the melting temperature is passed, the fluidity of phospholipid membranes is enhanced. The emergence of the transition between ordered and disordered phases is correlated to the specific molecular characteristics of the considered phospholipid. In particular, for the main component of the LS, i.e., the 1,2-Dipalmitoyl-sn-glycero-3 phosphatidylcholine (DPPC), the transition occurs around 41 ◦C, whereas the transition temperature can drop down to values below 0 ◦C for membranes of phospholipid having unsaturated acyl chains. Furthermore, the presence of cholesterol is essential for modulating the lipid packing. On the other side, when interfacial films, i.e., monolayers, are considered, the modulation of the phase behavior is commonly associated with surface pressure changes Π = γ<sup>0</sup> − γ, with γ<sup>0</sup> and γ being the surface tension for a pristine water/vapor (around 72 mN/m at 37 ◦C), and the surface tension of the monolayer, respectively. Therefore, if the interfacial density of phospholipids at the interface is low, it can be assumed that the monolayer behaves as a gas-like system, with the increase of the interfacial density pushing the monolayer through phases with higher lateral order, where the lipid mobility starts to be hindered, e.g., liquid expanded phase (LE) or liquid condensed phase (LC), to reach a 2D solid-like conformation for high values of Π [93].

Moreover, lipid membranes can undergo 3D assembly processes in aqueous medium to form a broad range of structure with lamellar and non-lamellar ordering. It should be noted that the specific molecular characteristics of lipids, and in particular their hydrophiliclipophilic balance (HLB), are essential in the modulation of the LS adsorption at the liquid/gas interface and reorganization during breathing [72]. Figure 2 show a sketch of different lipid structures that can emerge in LS under physiological conditions.

**Figure 2.** Different structures emerging in LS. Reprinted from Castillo-Sánchez et al. [72], with permission under Open access CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ (accessed on 20 December 2021).

The lipid polymorphism in LS surfactant plays a very important role for ensuring the correct respiratory function, which requires the fulfillment of three conditions: (i) efficient interfacial adsorption in a small time-scale; (ii) surface tension reduction down to very low values under compression without any weakening of the mechanical stability, and (iii) efficient respreading during expansion, ensuring the stabilization of the surface of the alveoli under recurrent compression-expansion cycles [72].

*De novo* secreted LS undergoes a continuous adsorption to the gas/liquid interface for guarantying the normal respiratory function. This entails that lipids are incorporated into the interface following a process governed by LS composition, and lipid concentration and structure, with the transition bilayer-to-monolayer being essential for the formation of functional interfacial films (see Figure 2). This requires the formation of non-lamellar structures at specific locations with the contribution of anionic lipids and SP-B and SP-C proteins [92,94–97]. In particular, SP-B protein presents a very important role in the first contact of LS with the interface, and also promotes membrane aggregation, fusion, and permeabilization, which may be related to the rapid lipid exchange between the interfacial film and the fluid phase underneath, commonly enriched in the less surface-active components, forming the so-called reservoirs [86,87,98–102].

The adsorption and spreading of the LS at the gas/liquid interface leads to the formation of an interfacial film which leads to a strong decrease of the surface tension during compression (at exhalation), minimizing the respiratory work [22]. Experimental evidences shown that the absorption of LS at water/vapor interface leads to an immediate drop of the surface tension from 70 mN/m to a value around 25 mN/m, and this latter value can be furtherly reduced to an almost negligible one upon a change of the interfacial area in the range

10–15%, which is equivalent to the final steps of the exhalation. This is possible because the composition of the interfacial film can be remodeled (squeezing-out process) to reduce the surface tension during exhalation [103], which requires excluding those molecules from the interface which cannot attain high values of surface pressure (unsaturated phospholipids, cholesterol, and surfactant proteins), a film enriched in DPPC remaining at the interface. This can lead to the formation of rather solid structures under high lateral pressures as those occurring in the alveolar surface during exhalation. This increases the lung compliance, and stabilizes the alveolar volume, which in turn contributes towards prevention of the premature alveolar collapse during exhalation [23–25]. Simultaneously to the compositional remodeling, the interfacial film is bound through protein-mediated interactions to reservoirs containing excluded lipids, proteins, and freshly secreted LS [92]. The association of the reservoirs with the interface plays a central role in the new remodeling of the interfacial composition occurring during inspiration by supplementing new molecules to the interface [102,104–109]. Furthermore, these types of structures contribute to the mechanical stability as a result of the high cohesion induced by protein-protein and protein-lipid interactions [87,104]. Therefore, disaturated lipids drive the surface tension decrease during exhalation, with surface-active proteins (SP-B and SP-C) acting as helper systems for ensuring such minimization of the surface tension.

The lateral organization of the LS interfacial film is reminiscent of a coexistence between two liquid phases, where domains enriched in DPPC appear surrounded by a disorder lipid phase. These domains grow during exhalation leading to a solid structure at maximum compression [92], which is reverted during inspiration as result of the quick replenishment of the gas/liquid interface by the adsorption of lipids from the reservoirs. This leads to the increase of the surface tension leading to the formation disordered phase. Therefore, the continuous remodeling of the LS films modulated by the surface proteins SP-B and SP-C connecting the interfacial film with the reservoirs ensures effortless breathing [32,72,110]. Figure 3 present a sketch showing the different phenomena occurring during the compression-expansion cycles of alveolar cavity, indicating the remodeling processes and their effect on the surface tension.

**Figure 3.** Sketch of the different remodeling processes occurring during compression-expansion cycles of the alveolar cavity. Reprinted from Da Silva et al. [32], with permission from Elsevier, Copyright 2021.

A last important process associated with the interfacial activity of the LS is related to the recycling of lipids and proteins excluded from the interfacial layer as result of the activity of alveolar macrophages and type II pneumocytes [111]. In particular, the SP-C protein presents a very central role in the recycling process contribution to the formation of small vesicles that can be easily taken up [90]. On the other side, SP-B stimulated the secretion of lamellar bodies in type II pneumocyte [112], whereas SP-A contributes to the mechanism regulating the inhibition of LS secretion when their concentration is enough in the alveoli [113].
