## 4 Discussion  
The alarming phenomenon of antimicrobial resistance has strongly highlighted that the use of antibiotics to treat upper respiratory tract-related diseases, such as OM, may not be resolutive. Currently, the annual incidence of acquired cholesteatoma is in the range of 9.0-12.6 cases per 100,000 adults and 3-15 cases per 100,000 children [3; 4; 28; 29; 30]. The topical treatments are unable to provide a complete cure of OM; rather, they are used to control preoperative infection or inflammation and to reduce the risk of postoperative complications [31; 32]. To avoid surgery, patients and otologists place great hope in new biotechnological treatments. Bacterotherapy, which relies on the administration of probiotics to compete with the infectious pathogens, has been recently emerging as an attractive option in this scenario [33]. This study aimed to harness the potential of bacterotherapy by developing an SA-based probiotic-laden delivery system. The combination of electrospray SA microparticles and electrospun SA/PEO ultrafine fibers served as a supportive substrate for topical application to the tympan membrane to address middle ear infections. In this research, SA was chosen as a suitable material to obtain the desired formulations. SA, a linear polysaccharide derived from algae, possesses advantageous properties, including high water-retention capacity, biocompatibility in many body settings, non-immunogenicity and tunable mechanical properties [34; 35; 36]. SA solutions can form physically crosslinked gels  
Figure 10: Ability of probiotic-loaded microparticles, visible as a white spot in the center of the plate, to inhibit _E. coli_ growth. (**A–C**) Example photographs showing microparticles on plates covered by _E. coli_: (**A**) inhibition zone (i.e., clear halo) following 24 h incubation of the probiotic-loaded microparticles; (**B**) inhibition zone following 72 h of incubation of the probiotic-loaded microparticles; (**C**) control plate “Crtl”. In which probiotic-free microparticles were added. (**D**) Bar graph showing mean values of the diameters of the inhibition zones \(\pm\) standard error of the mean. The diameters of the unloaded spots, barely visible, were approximately 10 mm (arrow in (**C**)). ***\(p<0.001\), ANOVA test. in the presence of divalent ions like Ca\({}^{2+}\), thus enabling the creation of solid structures that can be ideal for probiotic-laden solutions, by avoiding their exposure to potentially harmful conditions, like toxic chemicals or UV radiation [36]. The use of the electrospray technique allowed the production of consistent batches of SA microparticles, both probiotic free and probiotic-laden. These microparticles exhibited spherical or slightly elliptical shapes with narrow diameter distributions, measuring in the hundreds of microns (i.e., 346.5 \(\pm\) 10.0 \(\upmu\)m and 395.5 \(\pm\) 23.5 \(\upmu\)m for probiotic-free and probiotic-laden microparticles, respectively). Electrospray mild operating conditions hold great potential for encapsulating live cells within biopolymeric networks, making it a viable method for targeted bacterial delivery to specific body sites. Notably, this approach aligns with recent studies that have successfully employed electrospray for the encapsulation of live bacteria, as seen in the work of [PERSON] et al., involving hydrogel microspheres encapsulating _Lactobacillus plantarum_[17]. The produced microparticles were demonstrated to be easily collected by filtration and to act as effective carriers for the gradual and sustained release of encapsulated agents through various administration routes. In our case, the produced microparticles exhibited outstanding stability and minimal biodegradation under simulated biological condition. These characteristics make them suitable for formulations that require the gradual and controlled release of encapsulated agents, particularly in the context of chronic diseases with a challenging healing process. Electrospinning is a simple technique widely used in the biomedical field [37].