## 3 Characterization of Food-Grade Systems
### Biological Evaluation
#### 3.2.2 In Vivo Studies  
Once the in vitro tests have been performed and the formulation to be tested in vivo has been selected, the next step is to select the most appropriate animal according to the disease to be studied. Most of the reviewed studies explore the pathogenesis of ulcerative colitis, the modeling of which can be done by the immune method or by chemical stimulation. The latter technique is simple, reproducible and inexpensive and is therefore among the most commonly used methods in research in this field [149]. Mice and rats are the laboratory animals most frequently used to study ulcerative colitis, but differences in gut length have been observed between these two species. Mice have a shorter intestine than rats, plus the latter show a classification more similar to humans. Another advantage of rats is that they are well subject to modeling to recreate disease models, so they are the main experimental animals [150]. Rats with a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model were orally treated with alginate and chitosan nanoparticles loaded with berberine hydrochloride with the aim of evaluating the anti-inflammatory efficacy of the systems. Physiological characteristics of ulcerative colitis such as weight loss, colon weight and length, colon mucosal damage, and spleen index were evaluated. An in vivo study showed that nanosystems in addition to reducing disease activity and colonic mucosal damage are able to improve immunological function [91]. A recently introduced model of ulcerative colitis that is similar to human ulcerative colitis in terms of weight loss, reduced colon length, epithelial ulceration, and inflammatory cell infiltration is that induced by SDS [151]. Mice with this model of ulcerative colitis were used to evaluate the efficacy of binary composite NPs of zein and caseinate loaded with isoliquiritigenin. The fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylimidotricarbocyanine iodide (DiR) was encapsulated in the NPs (DiR@NPs) to track colonic biodistribution in vivo. The study suggested that the DiR@NPs were efficiently transported across the intestinal barrier into colonic tissues and that the zein/caseinate shell protected the active molecule, delivering it specifically to the colon inflammatory site (Figure 18). Indicators of ulcerative colitis (colonic length, body weight, and DAI scores) were significantly improved following administration of encapsulated isoliquiritigenin. The excellent therapeutic efficacy was further confirmed by the reduction of both pro-inflammatory cytokine levels and neutrophil infiltration in the colonic mucosa, suggesting an advantageous clinical application of these nanosystems in the treatment of ulcerative colitis [144]. The same model of ulcerative colitis was used by Zhang et al. to evaluate the efficacy of a combined formulation for curcumin delivery. This formulation consisted of NPs of zein and hyaluronic acid embedded in hydrogel microparticles prepared from alginate and chitosan. Retention and bioadhesion to colonic tissues were demonstrated by fluorescence imaging after intragastric administration, where the multifunctional platform showed combined advantages over individual systems in delivering curcumin to the colon. Oral administration of the formulation was effective in inhibiting intestinal inflammation (via the TLR4/NF-kB pathway) and consequently alleviating ulcerative colitis symptoms in mice [152]. A mouse model with DDS-induced disease was also employed by Zu et al. to evaluate the efficacy of exosome-like nanotherapeutics produced from tea leaves in delaying the development of IBD and preventing colitis-associated colon cancer. Formulations were produced from small, medium and large tea leaves. Two fluorescent molecules, DiR and 3, 3'-dioctadecylocarbonocyanine perchlorate (DiO), were loaded into the nanomedicines to evaluate biodistribution after oral administration.