*3.2. Evaluation of Particles Incorporation into DPPC Monolayers*

The evaluation of the incorporation of particles into DPPC monolayers, and their potential impact on the lateral organization of the lipid layers, was done on the basis of the modifications associated with the presence of particles in the Π*-A* isotherms of DPPC monolayers. This is important because the isotherms provide information related to the phase behavior of the mixed monolayers, and how the lateral packing of DPPC monolayers and their mechanical characteristics are modified due to the particles' incorporation, with such aspects being relevant on the biological function of the lipid layers. Figure 2 reports Π versus the area per DPPC molecule, *A*, normalized to its initial value, *A*<sup>0</sup> (Note that this initial value *A*<sup>0</sup> was fixed as a reference in all the experiments in 98 Å2/molecule), after the lipid spreading, corresponding to DPPC monolayers containing particles in a broad range of particles' weight fractions, *x*p. Notice that the weight fractions of the particles spread onto the DPPC monolayers correspond to estimated doses in the 6–115 mg/mL range (assuming a realistic thickness for the interface of 10 nm), which are compatible with the values reported for the dose of deposited particles in the lung surfactant layer upon inhalation [49–51].

The isotherm for DPPC spread at the bare water/vapor interface presents the typical features reported for monolayers of this lipid [44,52–54]. At the highest value of the reduced area (gas and liquid expanded –LE– phases), a mild increase of the surface pressure with the increase of the interfacial density, i.e., with the *A*/*A*<sup>0</sup> ratio decreases, was found. This proceeds up to a threshold value of the interfacial density, which defines the onset of the coexistence region between LE and liquid compressed (LC) phases (LE–LC coexistence is reached). This coexistence region is characterized by an almost vanishing change of the surface pressure (pseudo-plateau) as the interfacial density increases, which is associated with the disappearance of the LE phase as the re-orientation of the DPPC molecules occurs driving the system to a more ordered phase (LC). Once the LE-LC coexistence is overcome (*A*/*A*<sup>0</sup> ~ 0.45), a sharp increase of the surface pressure with the increase of the packing density within the LE expanded and solid phases was found until the rupture of the monolayer occurs at the collapse surface pressure, Πc.

The incorporation of particles into the DPPC monolayers does not modify substantially the shape of the isotherm in relation to that found for the pure lipid at the water/vapor interface, with this being almost independent from the chemical nature of the particles and the *x*<sup>p</sup> value. However, the incorporation of particles into the DPPC monolayers leads to the emergence of two effects: (i) the shifting of the Π—*A*/*A*<sup>0</sup> isotherms to more expanded states, i.e., to higher values of the reduced area, and (ii) the modification of the collapse pressure of the monolayer, i.e., the maximum surface pressure

that a monolayer can reach before its rupture. The former aspect is evidenced from the earlier lifting-off of the LE phase in the mixed monolayers than in pure DPPC, which can be understood considering that particles take up a part of the area available for the reorganization of the lipid molecules [55]. This leads to a situation in which the monolayers upon particles incorporation behave in such a way which is reminiscent of a monolayer with a higher effective interfacial density of DPPC. Therefore, it is possible to assume that particles' incorporation induces excluded area effects in the DPPC monolayer, which are strongly dependent on both the chemical nature of the particles and the *x*<sup>p</sup> value. Such dependences, and in particular that on the chemical nature of the particles, give an indication that the excluded area effects alone cannot account for the changes of the behavior of DPPC monolayers due to the incorporation of particles. This makes it necessary to analyze the role of the interactions between the different components forming the mixed layer (particle–particle, lipid–lipid and particle–lipid) to obtain a complete picture of the influence of particles in the behavior of DPPC monolayers. Such interactions affect the lipid lateral packing, and the aggregation and distribution of the particles at the interface, leading to a different behavior than that expected for systems where only the role of the area exclusion is considered.

**Figure 2.** Π–*A*/*A*<sup>0</sup> isotherms for DPPC Langmuir monolayers upon the incorporation of different weight fraction of particles at the interface (*x*p): CB (**a**) and SiO (**b**). Each curve corresponds to DPPC monolayers with a different weight fraction of particles at the interface (*x*p): (Δ) 0.00, (Δ) 0.10, (Δ) 0.33, (Δ) 0.75, and (Δ) 0.90.

The results show different dependences on the *xp* value for DPPC monolayers upon the incorporation of particles with different chemical nature. The increase of the amount of SiO2 particles incorporated into the DPPC monolayer shifts the isotherm to higher values of *A*/*A*0, which is explained as result of enhanced importance of the excluded area effects with the increase of *x*p. However, the situation is different when CB particles are concerned. The incorporation of CB particles into DPPC monolayer leads to two different regimes of behavior as a function of *x*p–on the impact of CB on the behavior of DPPC monolayers—were found: (i) for the smallest values of CB weight fraction, a strong shift of the isotherm to higher values of *A*/*A*<sup>0</sup> than those corresponding to pure DPPC monolayer was found as result of the area exclusion effect, and (ii) for the highest CB amounts at the interface, even though particles' incorporation leads to excluded area effects, its importance is decreased as *x*p increases.

The above-mentioned differences suggest the existence of different distributions for SiO2 and CB particles upon incorporation into DPPC monolayers. Thus, whereas the SiO2 particles may be incorporated into the DPPC monolayer as pseudo-2D aggregates which tend to occupy the maximum area available at the interface, the incorporation of CB particles leads to the formation of 3D particle-stacking with the increase of *x*p. Thus, the incorporation of CB particles in concentrations above a threshold value of *x*p leads to their stacking onto the preformed mixed monolayer, which may result in the formation of out-of-plane structures, such as wrinkles, folds, or buckles. This leads to a situation in which the effective concentration of particles at the interface is lower than that expected from the complete spreading of the particles within the area available, and, as matter of fact, to a reduction of the importance of the excluded area effects with the increase of *x*p [56–59]. The differences in the behavior of the DPPC monolayers upon the incorporation of SiO2 and CB particles are explained considering the different chemical nature of the particles. Thus, even though both types of particles are hydrophobic, the presence of dissociated silanol groups onto the surface of the SiO2 particles may introduce a repulsive electrostatic interaction between the particles, which facilities its dispersion within the DPPC films. However, when CB particles are considered, a strong hydrophobic attraction should be expected, which favors their agglomeration with the increase of *x*p [60].

The above discussion shows the strong impact of the chemical nature of the particles in their incorporation into DPPC monolayers, and its role in the excluded area effects. However, a complete picture of the impact of the particles on the DPPC monolayer behavior also needs a closer look at the the role of the interactions. For this purpose, a simple geometrical consideration may be useful. Assuming the incorporation of spherical particles which can cover a maximum area of the water/vapor interface defined as *N*π*r*2, with *r* being the radius of a single particle and *N* the number of particles incorporated into the monolayer, it would be expected that the fraction of interfacial area occupied by particles oscillate between a value lower than 1% for the lowest value of *x*<sup>p</sup> and a value around 10% for the highest one. However, the results show a higher impact than what was expected on the basis of the above simple geometrical considerations, with area expansions in the ranges 20–50% and 5–25% for SiO2 and CB particles, respectively. Thus, it is possible to assume that that the impact of particles in the lateral packing of DPPC monolayers results from a complex interplay between the excluded area effects, steric hindrance, and different types of interactions. The role of the interactions is clear from the analysis of the Π<sup>c</sup> dependence on *x*<sup>p</sup> shown in Figure 3. The results show that the incorporation of particles decreases the maximum surface pressure that the DPPC monolayers can reach before its rupture. This decrease of Π<sup>c</sup> indicates an irreversible incorporation of particles into the DPPC monolayers, which results in a reduction of the ability of DPPC to form highly condensed phases. This is in contrast with what is found when the incorporation of hydrophilic particles in DPPC monolayers is concerned; in those cases, an effective refinement of the interfacial composition is generally found for the highest compression degree, with a partial squeezing-out of the particles from the interface [24,61].

The decrease of Π<sup>c</sup> with the increase of *x*<sup>p</sup> results from the impact of particles on the lateral cohesion interactions of the molecules within the interface. Thus, the incorporation of particles, independently of their chemical nature, reduces the strength of the cohesion interactions between the lipid molecules as a result of the emergence of heterogeneities on the lateral organization within the film. The higher decrease of Πc, found when SiO2 particles are incorporated into the DPPC monolayer, in relation to those cases in which the incorporation of CB is considered, is explained considering

the differences of the steric hindrance associated with the presence of each type of particles at the interface [62]. Thus, whereas SiO2 particles tend to occupy the maximum area available at the interface, 3D stacking of particles are expected for CB particles, which results in a lower occupancy of the interface by CB particles. This leads to a situation in which SiO2 particles modify strongly the lateral organization of the lipid layer.

**Figure 3.** Dependence on *x*<sup>p</sup> of the collapse pressure, Πc, of DPPC monolayers upon the incorporation of CB () and SiO2 (•) particles. Note that the lines are guides for the eyes.

Additional information related to the incorporation of particles into the DPPC films are obtained from the changes of the quasi-static dilational elasticity ε<sup>0</sup> obtained from the isotherm as

$$
\varepsilon\_0 = -A \left( \partial \Pi \!/ \partial A \!/ \right) \Gamma. \tag{5}
$$

Figure 4 shows the surface pressure dependence of ε<sup>0</sup> for DPPC monolayers after the incorporation of different weight fractions of particles (data for the incorporation of CB and SiO2 are shown in panels a and b, respectively). The results show three different features when the elasticity of monolayers of pure DPPC are considered: (i) an increase of the elasticity up to a first maximum associated with the formation of the disordered LE phase, which presents a weak lateral packing (for the lowest values of Π), (ii) a drop of the elasticity, with the increase of Π, down to reach a quasi-null value for the LE–LC coexistence, and (iii) an increase of the elasticity within the LC phase up to reach its maximum value associated with an enhanced lateral packing of the monolayer, and then a drop of the elasticity as the monolayer approaches the collapse.

The incorporation of particles modifies dramatically the elasticity of the DPPC monolayers, with the average elasticity of the monolayer decreasing upon the incorporation of particles. This reduction of the monolayer rigidity is associated with a worsening of the lateral packing of the lipid molecules at the interface as a result of the weakening of the lateral cohesion interactions within the monolayer. A more detailed analysis of the impact of the particles' incorporation into the organization of the lipids within the interface is obtained from the changes of the quasi-static dilational elasticity corresponding to the maximum values of the elasticity for the LE and LC phases and to the LE–LC coexistence with *x*p (see Figure 5).

**Figure 4.** Quasi-static dilational elasticity ε<sup>0</sup> dependences on the surface pressure Π for DPPC Langmuir monolayer upon the incorporation of different weight fraction of particles at the interface (*x*p): CB (**a**) and SiO2 (**b**). Each curve corresponds to DPPC monolayers with a different weight fraction of particles at the interface (*x*p): (Δ) 0.00, (Δ) 0.10, (Δ) 0.33, (Δ) 0.75, (Δ) 0.90 and (Δ) 1.00. Notice that the lines are guides for the eyes.

**Figure 5.** (**a**) Dependences on *x*<sup>p</sup> of the maximum values of ε<sup>0</sup> for the LE (CB () and SiO2 (•) particles) and LC (CB (-) and SiO2 (-) particles) phases; (**b**) dependence on *x*<sup>p</sup> of the maximum values of ε<sup>0</sup> for the phase coexistence region (CB () and SiO2 (•) particles). Notice that the lines are guides for the eyes.

The absence of any noticeable change of the elasticity corresponding to the LE phases provide evidence that the lateral organization of the DPPC monolayer within this phase is not significantly modified, neither upon the incorporation of SiO2 particles nor after the incorporation of CB ones. This may be understood considering that the LE phase is an intrinsically disordered phase in which the role of the lateral van der Waals interactions between the lipids molecules is almost negligible, thus it may be expected that a slight modification of such interactions due to the inclusion of the particles does not modify significantly the lateral packing of the lipids within the LE phase. However, a closer look at the elasticity dependences for the LE phases provides evidence of a slight increase of ε<sup>0</sup> with *x*<sup>p</sup> as result of the incorporation of SiO2 particles, whereas the incorporation of CB results in an initial increase of ε<sup>0</sup> with *x*<sup>p</sup> up to a maximum for a *x*<sup>p</sup> value about 0.10, which is followed by a decrease of ε<sup>0</sup> down to a value slightly higher to that corresponding to pure DPPC. Thus, even though the impact of the particles is very limited in the LE phase, a certain degree of disorder is expected considering the experimental dependences, which is correlated to the differences in the particles' organization as function of their chemical nature. On the other side, the elasticities for the LE-LC coexistence and LC phases are strongly modified in relation to those corresponding to the DPPC monolayers. The incorporation of both types of particles reduces the maximum lateral packing of the monolayer, i.e., the quasi-static dilational elasticity for the LC phase decreases, independently on the nature of the particles. However, the impact of the incorporation of SiO2 particles is again stronger than that found when the incorporation of CB ones is considered. The effect of particles in the ε<sup>0</sup> value of the LE–LC coexistence results in being more intriguing, whereas the incorporation of CB particles into DPPC monolayers does not modify significantly the phase coexistence, and a strong increase of the elasticity of such region is found with the increase of *x*<sup>p</sup> for SiO2 particles. This allows one to assume that the impact of particles in the lateral packing of DPPC is driven by a complex balance involving different contributions, including the interactions involved in the mixed monolayers (hydrophobic vs. electrostatic), and the chemical nature and wettability of the particles (hydrophobicity vs. hydrophilicity of the particles). This leads to a hindering of the phase coexistence when SiO2 particles are incorporated as evidenced in the BAM images shown in Figure 6.

**Figure 6.** BAM images of DPPC monolayers upon the incorporation of CB and SiO2 particles at two different values of *x*p for surface pressure about 7.5 mN/m, corresponding to the LE–LC phase coexistence.

The BAM images show that, whereas the DPPC monolayers upon the incorporation of CB particles presents ellipsoidal-like domains which are similar to those found for pure DPPC monolayers, the incorporation of SiO2 particles leads to the disappearance of such domains, i.e., the incorporation of SiO2 drives to a hindering of the LC domains formation, which is compatible with the increase of the quasi-static dilational elasticity corresponding to such region. Furthermore, the BAM images also make clear the different distribution of the particles as a function of their chemical nature. Thus, a decrease of the size of the bright spots associated with particle agglomerates was found when CB particles are considered. However, no bright spots are found when the incorporation of SiO2 is analyzed for the lowest value of *x*p, which provides evidence that the distribution of the particles within the interface is better.
