*2.2. Monolayers Preparation*

The lipid monolayers were prepared at the water/vapor interface by dropping controlled volumes of DPPC from its solution in chloroform (concentration about 1 mg/mL or 1.36 mM) using a high-precision Hamilton syringe (Hamilton Company, Reno, NV, USA). This methodology ensures the control of the interfacial density of DPPC, Γ, upon solvent evaporation. The initial interfacial density of DPPC spread at the water/vapor interface Γ<sup>0</sup> was fixed in all the experiments in a value of 1.7 μmol/m2, corresponding to an area per molecule of about 98 Å2.

The preparation of the mixed monolayers was done following a two-step approach: (i) a DPPC monolayer was obtained from the spreading of the lipid from its solution in chloroform (concentration 1 g/L) at the bare water/vapor interface, and (ii) a given amount of the particle dispersion (concentration 1 g/L) was spread onto the preformed DPPC monolayer, again using chloroform as the spreading solvent (Notice that particles dispersions were sonicated during 15 min using a laboratory ultrasound bath; this allows for minimizing particles aggregation before their spreading). This procedure allows for obtaining monolayers with specific DPPC: particles weight ratio at the interface. Once monolayers including particles and DPPC are obtained, it is necessary to wait during1hbefore starting the experiments to ensure the complete evaporation of the solvent and, in the case of mixed monolayer, the achievement of the equilibrium of the composite system which is driven by the nanoparticle–lipid interactions [21,23]. It is worth noting that the monolayers studied here cannot be a strictly considered mixed monolayer because they were not obtained after the spreading of a mixed dispersion containing the DPPC and the particles (first, the DPPC is spread at the pure water/vapor interface, and then the addition of the particles is made onto the preformed DPPC monolayer). However, for the sake of simplicity, the term mixed monolayers will be used for monolayers involving the DPPC and the particles.

The temperature was fixed 22.0 ± 0.1 ◦C in all the experiments. Even though this temperature is far from the physiological one (37 ◦C), the main conclusions obtained in our study are extrapolated, at least from a semi-quantitative perspective, to the physiological conditions. This is because the phase transition of DPPC appears above the physiological temperature, thus only a shift of the phase behavior with the temperature is expected, without any significant impact on the main physico-chemical insights extracted from the experimental results [41].

It is worth mentioning that the properties of the mixed monolayers obtained upon chloroform evaporation will be affected by the conditions in which DPPC and particles are mixed [30]. This work has used a methodology for the preparation of the mixed layers where the interaction between particles and lipid layers occurs only at the water/vapor interface. This may be considered similar to that which happens during the interaction between environmental pollutants and lipid layers. However, this study does not consider two aspects that may have impact when in vivo conditions are concerned: (i) the presence of chloroform during particles addition may affect both the lateral packing of the DPPC molecules and the DPPC–particle interactions [30], and (ii) the interaction of particles and the lipid layers may be affected for specific mass transport boundary conditions which cannot be included in our studies using Langmuir monolayers [42,43].
