2.3.2. DLS Analysis of Emulsified Dispersion of Lipomatrix in FaSSIF-V2

The DLS analysis of the sample containing SRO entrapped in Lipomatrix and dispersed in FaSSIF-V2 produced the size distribution curve reported in Figure 6 (red line) whose descriptive parameters are reported in Table 2.

**Figure 6.** Particle size distribution of FaSSIF-V2 (black Line) and LIPOMATRIX in FaSSIF-V2 (red line) after sonication (*n* = 5).



The FaSSIF-V2 sample shows a high polydispersity index (0.703) with three well-defined particles populations (769, 161 and 5468 nm) of which the first one is the most abundant and the third the least one. The sample of Lipomatrix dispersed in FaSSIF-V2 shows an elevated polydispersity index (0.529) as well, even though lower than FaSSIF-V2 alone and is composed of three groups of particles (528, 27, and 4511 nm), of which the first one is largely the most represented. The DLS evaluation seems to consolidate the visual hypothesis that Lipomatrix can effectively disperse SRO in FaSSIF-V2, giving rise to an even more finely dispersed system than FaSSIF-V2 alone (lower polydispersion index and presence of a prevalent population around 500 nm). Lipomatrix seems therefore to create a more uniform micellar dispersion in force of the presence of both ionized ASP and MDGFA. ASP becomes partially ionized in the presence of enteric fluids (pH > pKa) and in force of this ionization it behaves as hydro-soluble surfacting agent, capable of emulsifying SRO in synergy with MDGFA and bile salts, generating mixed micellar structures [28,29] (Figure 7). This data gives an analytical confirmation of the visual macroscopic difference of emulsification capability of SRO entrapped in Lipomatrix compared to the same unformulated oil dispersed in FaSSIF-V2 as described in Section 2.3.1. Finally, the comparison of the two specimens shows that a new population ascribable to Lipomatrix, with average hydrodynamic diameter of 27 ± 6 nm, was generated.

**Figure 7.** Representation of Lipomatrix mechanism of delivery: lipophilic molecule entrapped in a mixed micellar structure composed of MDGFA, ASP, and bile salts.
