## 3 Characterization of Food-Grade Systems
### Biological Evaluation
#### 3.2.1 In Vitro Assays
To understand if the superior performance of the coated CS-Zein NPs could be attributed to increased cellular uptake, the fluorescent probe coumarin-6 was encapsulated in the NPs (C6@CS-Zein NPs). Flow cytometry and confocal laser scanning microscopy (CLSM) confirmed the hypothesis that chondro sulfate coating enhanced CD44 receptor-mediated endocytosis and consequently the cellular uptake (Figure 17) [143]. These two cell lines were also used by Xiao et al. to evaluate the cellular uptake of zein and caseinate NPs loaded with isoliquiritigenin for the treatment of ulcerative colitis. For the in vitro study, NPs were labeled with coumarin-6, and the results of CLSM analysis showed that the encapsulated molecule showed significantly higher cellular uptake compared to the free molecule on both cell lines [144]. Another cell line frequently investigated to assess the absorption of orally administered drugs is the Caco-2 cell line [145]. In a study by Salah et al., Caco-2 cells were stimulated with TNF-\(\alpha\) and the anti-inflammatory effects of the formulations were observed following early (stimulus + treatment) and late (stimulus for 24 h followed by treatment) treatments. Although the in vitro permeability study demonstrated effective release of encapsulated curcumin across the epithelial barrier, to obtain more reliable results the authors also investigated absorption on an ex vivo murine colon explant [90]. Figure 17: Fluorescence images showing cellular uptake of free C6, C6@CUL-Zein NPs, C6@CS-Zein NPs, and C6@CS-Zein NPs (with 2 \(\upmu\)g/mL CS pretreatment) in NCM 460 and raw 264.7 cells for 4 h. Scale bar is 50 \(\upmu\)m. Adapted with permission from Ref. [143]. 2021, American Chemical Society. Inflammation is a process that involves several cell layers and should be considered to improve the predictivity of the results. To accurately simulate the architecture of the human intestinal epithelium, an innovative triple co-culture model was developed that included Caco-2 cells, HT29-MTX cells and raji-B cells. The lack of ability to generate mucus is a disadvantage of the Caco-2 cell line, so HT29-MTX cells derived from calyciform cells were useful to simulate a more predictable model experimental cell [146]. The Raji B cell line, derived from a human Burkitt's lymphoma, was used to induce differentiation of Caco-2 cells into M cells of reduced enzyme activity compared with Caco-2 cells in monoculture [147]. This interesting model of in vitro intestinal permeability was employed by Prezotti et al. which prepared mucoadhesive beads of gellan gum for colon-targeted administration of RSV. The permeability results showed that the mucoadhesive carriers significantly reduced the permeability of this compound and prolonged contact with colon cells enhanced its local targeting. Therefore, the triple co-culture model proved to be useful for an accurate reproduction of RSV transport through cell monolayers [148]. Although in vitro models do not consider the cellular homeostasis that governs multiple factors in vivo, they are necessary assays to establish biocompatibility and preliminarily screen the most promising formulations to be tested for in vivo applications.