## 3 Results and Discussion
### In Vivo Toxicity Study on Nasal Mucosa  
The investigated nanocarriers were subjected to a toxicity study to evaluate their safety towards nasal tissues [50,51]. A first control experiment was performed to investigate the potential adverse effects on mouse nasal mucosa of unloaded EUD NPs (EUD3A). NP suspension (50 \(\mu\)L) was administered intranasally for \(1\) week. The results of the histological analysis are shown in Figure 5.  
Figure 4: DSC thermograms of RS, RL1, RS/RL physical mixture (p.m.), DIC, DIEP, RS/RL-DIC p.m., RS/RL-DIEP p.m., EUD3A, EUD3A_DIEP3, and EUD3A_DIC4.  
Administration of EUD3A had no effect on the normal morphology of the nasal mucosa. A subsequent experiment was performed to investigate the effects on mouse nasal mucosa of EUD3A_DIC4, EUD3A_DIEP3 in comparison with native DIC and DIEP. Suspensions of the different formulations (50 \(\mu\)L) were administered intrananally (30 mg/kg b.wt. dose) for 1 week. The results of the histological analysis revealed that administration of EUD3_DIC4, EUD3A_DIEP3 had little effects on the surface columnar epithelium and muciparous glands of the nasal mucosa, the only detected abnormalities being clusters of mucus and shed epithelial cells in the nasal cavities and sporadic dilatation of mucosal blood vessels. Administration of EUD3A_DIC4, EUD3A_DIEP3, or free DIEP did not result in appreciable abnormalities of the different histological components of the nasal mucosa.  
Conversely, DIC-induced secretory activation of muciparous glands, as judged by reduced size of their adenomeres and occurrence of clusters of mucus embedding shed epithelial cells within the nasal cavities, as well as rarefication of cilia in the columnar surface epithelium. Moreover, DIC also caused prominent and diffuse dilatation of blood vessels in the mucosal lamina propria.  
Specifically, the septal mucosa showed normal features in the mice given EUD3A, while it evidenced marked blood vessel dilatation (arrowheads) in those given DIC. The muciparous glands (central panels, asterisks) showed normal size and appearance upon treatment with EUD3A, while they are reduced in size, as occurs during secretory activation, upon treatment with DIC; membranes of mucus and shed epithelial cells can also be  
Figure 5: Histological features of the nasal mucosa of mice administered with the control formulation of unloaded EUD NPs (EUD3A); neat diclofenac (DIC) and diclofenac epolamine (DIEP); and EUD NPs with loaded with DIC or DIEP (EUD3A_DIC4 and EUD3A_DIEP4). Hematoxylin and eosin; bars = 100 \(\mu\)m.  
seen within the nasal cavities (arrows). The ciliated columnar epithelium showed normal features upon treatment with EUD3A, while it displayed rarefication of cilia upon treatment with DIC.  
The septal mucosa shows normal features in the mice given EUD3A_DIEP3 and DIEP, while it shows clusters of mucus and shed epithelial cells within the nasal cavities (arrows) in those given EUD3A_DIC4. The muciparous glands (central panels, asterisks) show substantially normal features and size with all the treatments. The ciliated columnar epithelium (right panels) also shows normal features with all the treatments. Occasionally, marked blood vessel dilatation (arrowheads) was detected in the nasal mucosa of mice given EUD3A_DIC4.  
Overall, both formulations do not cause any irritation or toxicity compared to the free DIC, this might be due to the encapsulation of the drug within the polymeric system, thereby reducing its direct irritant effect on the mucosal tissue, and can be considered to be biocompatible for nasal administration.