## 3 Results and Discussion  
### Preparation of Blank Eutraggit NPs  
The EUD NP batches reported in Table 1 were produced by the QESD method [25; 26; 27; 28] to assess the effect of formulation variables on their technological properties.  
In particular, the effect of copolymers concentration and the stirring speed during the preparation were evaluated.  
PCS analysis revealed mean particle size in the range between 285 and 380 nm and a narrow width of distribution (PdI \(\leq\) 0.25). Among the experimental variables tested, copolymer concentration did not seem to affect the particles size response; conversely, a higher stirring speed was associated to the formation of relatively larger NPs, although the differences between a 5000 rpm speed (producing NPs of about 300 nm) and the 15,000 rpm one (with NP sizes between 350 and 380 nm) were very limited. In the QESD method, the stirring speed influences the velocity by which the dropping organic phase, which contains the polymeric material, is dispersed into the aqueous one forming a 'quasi-emulsion' of polymeric droplets. The formation and following precipitation of the NPs from these droplets derives from the counter-diffusion of the organic solvent towards the aqueousphase and of the water inside the polymer organic solution [25; 26]. Therefore, experimental evidences would support the idea that at a lower stirring speed, the velocity of counter-diffusion of the two liquid phases prevailed on the dispersion of the organic (polymeric) phase in the aqueous one, with a quicker formation of relatively smaller particles.  
Based on the above trials, the polymeric systems EUD3A, that resulted to be the most homogeneous formulation (PdI 0.119 \(\pm\) 0.06) with a mean size \(<\) 300 nm was selected and loaded with the chosen active compounds, namely DIC and DIEP, producing the NP batches EUD3A-DIC and 3A-DIEP, respectively (Table 2). On this system, the effects of different drug-to-polymer ratios (from 1:2 to 1:4 for DIC; from 1:2 and 1:3 for DIEP) on NPs mean size and PdI were further evaluated.  
Both the actives showed a certain difficulty to be encapsulated in the polymer matrix; in fact, DIC shows a sparingly solubility in ethanol (25 mg/mL), whereas DIEP, although is much more soluble in the organic solvent, when came into contact with the aqueous phase produced some stiffing material; for this reason, DIEP-to-polymer 1:4 ratio was not further considered (Table 2).  
Furthermore, lower polymer concentrations in the organic phase were used to allow incorporating higher drug amounts. Results showed that the drug in the form of a free acid or as a salt significantly affect the physico-chemical properties of EUD-NPs: the DIC-loaded systems produced with a 1:3 or 1:4 drug-to-polymer ratio (EUD3A_DIC3 and EUD3A_DIC4) had mean sizes around 300 nm, with a good size homogeneity (PdI \(<\) 0.4). In the presence of a lower drug/polymer ration (EUD3A-DIC2) larger nanoparticles were produced, with a heterogenous particle population. Using DIEP, particles with a size of 1434.3 \(\pm\) 0.34 (EUD3A_DIEP3) and 1908.1 \(\pm\) 0.51 (EUD3A_DIEP2) were obtained, always associated to a relatively low PdI value (PdI \(<\) 0.4).  
Formulations EUD3A_DIC4 and EUD3A_DIEP3 with notable dimensional differences were selected for further technological evaluations. Moreover, despite the low homogeneity of the EUD3A_DIEP3 batch, compared to EUD3A_DIC4, it could be an appropriate formulation for a local mucosal interaction after nasal administration, since particles with a size less than 500 nm could follow the trigeminal and/or olfactory pathways after IN instillation [30].