## 3 Results and Discussion
### Mucoadhesive Evaluation of Optimized Free and Drug-Loaded EUD NPs  
Several features may hamper drug absorption through the nasal mucosa, such as molecular weight and permeability. The amount of drug transported from nose to the systemic circulation can be quite variable, ranging from almost 100% to less than 1% of the administered dose [34]. Therefore, several strategies are under investigation to improve drug absorption after intranasal application [35]. The use of bioadhesive polymer is considered a convenient method to enhance drug permanence to epithelial surface and improve its diffusion to the systemic pathway, when needed. Polymers can be considered \"mucoadhesive\" if they possess a number of hydrophilic groups able to attach to mucus or the cell membrane by different ways, such as forming hydrogen bonding or by hydrophobic or electrostatic interactions. Polymers with hydrophilic \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Batch** & **Drug-to-Polymer Weight Ratio** & **\% Polymer (w/\(\underline{\underline{\underline{\underline{\underline{\underline{ \underline{\underline{\underline{\underline{\underline{\underline{\underline{\underline{ \underline{ \underline{ \underline{ }}}}}}}}}}}}}}\))** & **Z-AVE (nm) \(\pm\) SD** & **PdI \(\pm\) SD** \\ \hline EUD3A-DIC2 & 1:2 & 3 & 706.3 \(\pm\) 0.21 & 0.993 \(\pm\) 0.03 \\ EUD3A-DIC3 & 1:3 & 3 & 196.6 \(\pm\) 0.12 & 0.345 \(\pm\) 0.12 \\ EUD3A-DIC4 & 1:4 & 3 & 244.2 \(\pm\) 0.13 & 0.258 \(\pm\) 0.11 \\ EUD3A-DIEP2 & 1:2 & 3 & 1908.1 \(\pm\) 0.51 & 0.375 \(\pm\) 0.05 \\ EUD3A-DIEP3 & 1:3 & 3 & 1434.3 \(\pm\) 0.34 & 0.365 \(\pm\) 0.32 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Composition, mean size (Z-Ave) and polydispersity index (PdI) of DIC-loaded EUD NPs (EUD3A-DIC) and DIEP-loaded EUD NPs (EUD3A-DIEP). groups can also swell in water and thus expose the maximum number of adhesive sites available for interaction with the biological compartments [36]. The presence of quaternary ammonium groups imparts a positive charge to RS and RL copolymers, promoting their interaction with anionic drugs [26; 28] or the surface of some target tissues. The application of RL and RS copolymers for their cationic charge and uncoadhesive properties to provide a direct contact of NPs with the gastrointestinal tract (GIT) mucosa has been investigated, which may increase the likelihood of cellular uptake by endocytosis and increase the transit time of NPs in the GIT, leading to an improved therapeutic efficacy [37]. Herein, a preliminary evaluation of interaction with mucin, which is one of the main components of the nasal mucus layer, was carried out in order to assess the potential application of EUD NPs free or drug-loaded for IN administration. ZP measurement is a common method to investigate the uncoadhesive properties of several biopolymers and evaluate the interactions of NPs with mucin. As shown in Figure 1, EUD3A and EUD3A-DIC4 present a strong positive surface charge that was significantly converted toward lower values after incubation with mucin. These results suggest that electrostatic interactions occurred between the positively and negatively charged entities. Significant interactions occurred also for EUD3A_DIEP3 and mucin, despite EUD3A_DIEP is characterized by a negative charge, probably due to the partial absorption of the drug on particles surface. The principal process in the delivery and absorption of a drug by the nasal cavity route is through the mucus. Mucin is a protein that has the potential to bind with solutes and thus affect the diffusion process. Mucus is a viscoelastic gel that lines the lumen of the respiratory tissue. The sialic acid and sulphate content are very high in most of the moist mucosal epithelial interfaces, thereby imparting a pronounced negative charge [38]. Increasing the residence time of drug-loaded NPs in the packed layer of mucus enhances the chances to cross the mucus barrier and promote drug systemic diffusion [39].