*2.6. Pseudo-Semantic Analysis*

A new approach to evaluate membrane fluidity is to evaluate SCD by pseudo-semantic analysis. The SCD parameter, obtained from MD analysis, is converted into alphabetical strings, which are compared and clustered by similarity. The advantage compared to the previous analysis, is the possibility to compare the different membrane/probe systems and to analyze the fluidity of the individual frames during a simulation. The result, displayed through a dispersion graph, is shown in Figure 8.

**Figure 8.** t-SNE dispersion graph. Each point corresponds to a frame of molecular dynamics. Circle dimension is related to the time (ns). Green circles: POPC membranes; red/orange: DPPC-Chol systems.

In Figure 8, the six systems analyzed are shown. Each simulation of MD generates 11 frames, whose dimensions increase with the time of the simulation (ns). Each circle corresponds to one of the 11 frames of the trajectory. In different shades of green are shown the systems in POPC, and in red/orange the systems in DPPC-Chol. The analysis is capable to capture the similarity among different membranes at different times. Figure 8 shows that DPPC-Chol membranes are rather similar, forming almost a single cluster (top right). This fact indicates that the presence of the probe does not significantly perturb the membrane and that the order of the aliphatic chains during the whole dynamics remains similar. On the contrary, POPC membrane (and reasonably any kind of Ld membranes), are slightly influenced by the presence of the probe. The ordering effect of the probe is revealed by the change in the position of the POPC + T\* cluster toward the DPPC-Chol cluster.
