*6.1. Interaction of Particles and Lung Surfactant Films: A Matter of Size*

Particle size is a very important parameter controlling the interaction of particles with cells, affecting particle uptake. Furthermore, particle size affects their cytotoxicity and the induction of inflammatory response [215,216]. Therefore, it may be expected that size can also influence the interaction between inhaled pollutants and the LS film.

Dwidedi et al. [57] studied the interaction of hydrophobic poly(organosiloxane) particles with two different sizes (12 and 136 nm) with two LS models, and found that the smallest particles do not induce any significant modification of the phase behavior of DPPC layers, even though the morphology of the Lc domains undergoes a slight change. However, when the incorporation of the biggest particles was considered, the lifting-off of the surfacepressure isotherm was shifted to lower values of the compression degree, and the DPPC film undergoes a strong modification of its phase behavior. The situation changes when 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and the SP-C were used together with DPPC as model LS. In this case, it was again found that the smaller the particle size the smaller the modification of the phase behavior. However, the incorporation of big particles (diameter 136 nm) induces a strong modification of the interfacial phase behavior of the LS model. These modifications were found to be stronger at the highest surface pressures (above 35–40 mN/m), which is critical for the normal physiological function of the LS film, and in particular for the remodeling of the composition of the interfacial, inhibiting the incorporation of vesicles from the adjacent fluid phase to the interface. Furthermore, particles with a diameter of 136 nm lead to a fluidization of the interfacial film, i.e., reduce the elasticity of the LS film, independently of the model used. This may be explained considering that particle incorporation leads to a disruption of the lateral packing of the LS film, modifying the cohesion between the molecules in the monolayer. This results from the complexity of the interaction balance governing the interfacial packing, which includes different contributions, e.g., steric hindrance, excluded area effects, and other type of interactions [200]. This leads to a strong influence of the particle size and their concentration in the performance of LS film [118,119,133,179].

The effect of the particle size in the biophysical properties of LS films is strongly dependent on the specific chemistry of the probed particles. This is clear considering the existence of some studies in which the effect of particles is rather independent of their size, and emerges only dependent on the particle nature [173,178,217], whereas in other cases, size emerges as the main aspect governing the impact of particles with LS films.

Orsi et al. [178] reported that the modification of the phase behavior, lateral packing of the molecules at the interface and interfacial dynamics of DPPC films upon the incorporation of particles with size in the range 9–60 nm emerged rather independent of the specific particle size. Furthermore, the incorporation of particles into the DPPC films hindered the formation of condensed phase, leading to an interfacial organization that was reminiscent of what is expected for Pickering emulsions (2D Pickering emulsion-like structure) in which particles decorated with DPPC molecules are distributed around ordered domains of DPPC. This organization results in a reduction of the line tension of the domains in relation to that which is found for pure DPPC, and consequently their growth is hindered, i.e., the formation of domains with smaller size is found [179] in agreement with the molecular dynamic simulations by Curtis et al. [217]. Figure 8 presents a schematic representation of the structure emerging from the incorporation of hydrophilic particles into DPPC films.

Contrary to what was reported for the incorporation of hydrophilic silicon dioxide particles, Ku et al. [218] found that the incorporation of gelatin particles into DPPC monolayers leads to a modification of the interfacial behavior of the lipid in such a way that the resultant emerges strongly dependent on the specific dimensions of the incorporated particles. Furthermore, it was found that the largest particles (in this case with an average diameter around 236 nm) present the strongest interaction with the LS model layer altering both the surface pressure-area per molecule and the surface potential-area per molecule isotherms, and the reduction of particle dimensions weakens such interaction. Particle incorporation into DPPC pushes the phase behavior to more expanded states, making the rearrangement of lipid molecules at the fluid interface difficult.

**Figure 8.** Sketch showing the interfacial organization of interfacial films of DPPC upon the incorporation of hydrophilic silicon dioxide and experimental image of such organization obtained by epifluorescence microscopy. Reprinted from Orsi et al. [178], with permission under Open access CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ (2015) (accessed on 20 December 2021).

The above discussion evidences clearly that the influence of the size on the impact of particles in the physico-chemical properties of LS model is far from clear, and the current framework is controversial. An additional contribution to this controversy emerges from the study by Kodama et al. [219] in which the interaction of particles with different dimensions with a commercial LS formulation (Survanta) was explored. They found that only very small particles (average diameter about 20 nm) drive a significant modification of the interfacial phase behavior of the LS model. The multiple scenarios found for the interaction of particles and LS models can be understood considering that the effect of particles is dependent on a complex interplay of different factors, including the types of particles and LS model as well as the specific interfacial behavior of the model used.

It should be noted that together with particle size, there are physical parameters related to the particles, e.g., total surface area and specific surface area, chemical nature, and surface charge, that influence their impact on the behavior of LS layers. This is better understood considering that the alteration of LS behavior as a result of the particle incorporation emerges from two different directions: (i) particle aggregation in the LS layers and (ii) specific particle-LS interactions [219]. This perspective agrees with the results found when the interaction of particles in vivo is analyzed. The association of particles with LS films is strongly correlated to the specific nature of the interactions occurring within the system, and the ability of particles to be coated for an LS corona [220]. On the other side, the aggregation plays a very important role in the deposition of particles along the respiratory tract, as well as on the interaction with LS and clearance mechanism. It should be noted that together with the size, there are many other physico-chemical parameters of the particles modulating their interactions with LS [7,66].
