## 3 Characterization of Food-Grade Systems
### Biological Evaluation  
#### 3.2.1 In Vitro Assays  
In preclinical research, in vitro models play a crucial role in establishing the potential therapeutic effects before moving to in vivo experimentation. In addition, conducting extensive in vitro studies is of great importance to ensure successful formulation with less demanding techniques than animal experiment, both on the economic and ethical fronts. Culture techniques are used to study the cytotoxicity, permeability, and targeting efficacy of formulated systems [141]. Some examples of the reviewed articles are discussed below. Figure 15: _Cont._  
To alleviate the inflammatory disorders of ulcerative colitis, totally food-grade silk sericin (SS) nanocarriers loaded with proanthocyanidins (PAC), natural polyphenols with free radical scavenger activity, were fabricated. The in vitro study showed that the antioxidant activity of the nanosystems, in addition to being exerted by the polyphenols, was also exerted by the silk sericin matrix. The ferric reducing antioxidant potential (FRAP) assay showed that the combination of the two components synergized the antioxidant properties of the individual compounds in a concentration-dependent manner. Irritation of nanocarriers on mucous membranes was evaluated on a chorioallantoic membrane (CAM), which showed the absence of signs of irritation such as hyperemia, hemorrhage, and blood vessel coagulation even at the highest concentrations tested (Figure 16). Hemocompatibility was studied by observing hemolysis induced by samples incubated with fresh blood. Encapsulation of proanthocyanidins reduced the mild hemolytic activity that had been observed for free polyphenols. Consistent with the absence of irritation and hemolysis, the cytotoxicity of the formulations was confirmed by MTT assay  
Figure 16: Photographs of CAM before and after exposure to SS, PAC, and SS/PAC1. adapted with permission from Ref. [142]. 2022, Elsevier B.V.  
Figure 15: Destabilization kinetics expressed by Turbiscan Stability Index (TSI) of orange fruit juice (CTRL), empty cluster of nanoparticles (CL_NP), and coated Lacticaseibacillus rhamn GG (Coated_LGG) stored 7 days at 25 \({}^{\circ}\)C (**a**) and backscattering profiles (\(\Delta\)BS) of orange fruit juice (CTRL) (**b**), empty cluster of nanoparticles (CL_NP) (**c**), and coated Lacticaseibacillus rhamns GG (Coated_LGG) (**d**) stored in Turbiscan® at 25 \({}^{\circ}\)C for 1 week. Adapted with permission from Ref. [140], 2021, The Author(s). performed on RKO cells of human colorectal carcinoma. The study showed that cell viability was concentration dependent, although after 72 h of incubation even the highest concentrations tested indicated the absence of toxicity [142]. Another treatment for ulcerative colitis was proposed by Wang et al. The authors encapsulated magnolol in core-shell nanoparticles (CS-Zein NPs), consisting of a zein core and a chondro sulfate coating, which were in turn embedded in hydrogel microspheres by electrospray (ES). For comparison, the anionic polysaccharide sodium carboxymethyl cellulose (CUL) was used to replace chondro sulfate coating and to prepare untargeted NPs (CUL-Zein NPs). It is known that in ulcerative colitis, inflammation of lesions often occurs in normal colonic epithelial cells and that inflammatory factors are secreted by macrophages [101]. Therefore, normal human colon epithelial cells (NCM 460) and macrophages (Raw 264.7) were selected by the authors to examine cell viability and to determine the concentrations to be tested to assess the anti-inflammatory effect. To determine the anti-inflammatory effects, the two cell lines were stimulated with lipopolysaccharideacharide, and after the incubation period, the amounts of proinflammatory (TNF-\(\alpha\), IL-\(\beta\), IL-6) and anti-inflammatory (IL-10) factors in the cell supernatant were determined by ELISA. The results showed that chondro sulfate coating enhanced the anti-inflammatory effect.