## 3 Characterization of Food-Grade Systems
### Technological Characterization
#### 3.1.2 Surface Charge and Mucoadhesion  
Another critical feature of systems designed for colon targeting is the surface chemistry, which determines properties such as surface charge and mucoadhesion. These properties play an important role in the interaction of the systems with the gastrointestinal tract, as they affect mucosal penetration and tissue permeability, as well as the fate of the carriers in vivo [87]. The NPs surface could also be functionalized to ensure a site-specific response. For example, folic acid is a ligand of great interest for anticancer drug delivery, since cancer cells overexpress specific receptors for this molecule and ensure targeted action to the tumor site [88]. Carriers with mucoadhesive properties could improve interaction with the mucosal barrier and promote cellular absorption, which is an additional advantage for drug delivery in the treatment of colitis, where ulcerated walls produce a thicker mucus layer [89]. Generally, the mucus production is increased in IBD and in an inflammatory context the surface charge of the intestinal epithelium is overall cationic. With the goal of increasing mucoadhesion and epithelial retention time, many studies have focused on the production of carriers with anionic or neutral surface charges [90]. Although chitosan has frequently been used in oral delivery systems for colon-targeted delivery, the cationic mucoadhesive behavior of this polymer promotes adhesion to the upper gastrointestinal tract and impairs transport and targeting efficiency to the colon. Therefore, chitosan is often coated or combined with pH-sensitive polymers. An example is the totally food-grade mucoadhesive system prepared by Sun et al., consisting of sodium alginate and chitosan NPs (SA-CS NPs) loaded with berberine hydrochloride to treat the colonic ulcer lesions [91]. SA-CS NPs showed a size of 257 nm, negative charge (\(-\)37.9 mV) and mucopentrating ability. Such behavior was studied on rat colon mucus by fluorescence using Rhodamine B as a probe. For comparison, Authors used NPs made of chitosan alone (CS NPs) as a positively charged system. The small size of both NPs allowed them to pass through the pores (500 nm in size) of the mucin mesh structure, but the different surface charge affected the depth of penetration into the colonic mucosa. Indeed, positively charged NPs consisting of chitosan were retained in the superficial layer (0-200 \(\upmu\)m) of the mucosa that, being renewed every 5 h, made the clearance of the nanocarrier easier. Conversely, the negatively charged NPs consisting of CS and alginate showed a 2.9-fold higher mucus penetration capacity, with a penetration depth over 600 \(\upmu\)m, a positive behavior to ensure a longer permanence and accumulation of the carrier and drug in inflammation sites of the colon. Chitosan has also been used in combination with pectin for curcumin delivery in the treatment of colon cancer. Pectin has been used both to limit the premature release of curcumin in the gastric tract but also to confer mucoadhesive properties to the nanosystems at the colonic level. Mucoadhesion was studied by dispersing the systems in solutions of mucin type III from pig stomach at different concentrations and pH (1.2, 6.8 and 7.4). The change in pH affected the surface charge of both NPs and mucin. The mucoadhesive properties of pectin under alkaline conditions were well known, but the study showed that under these pH conditions the amine groups of chitosan were also ionized, establishing stronger polyelectrolyte bonds with the mucin that had assumed a more accessible \"rod-like\" structure. In addition, pectin in an alkaline environment underwent swelling resulting in premature release of curcumin, but the combination with chitosan ensured the formation of more stable systems whose degradation occurred in alkaline media enriched with pectinase to provide a localized therapeutic effect.