Santarcangelo, Cristina (2023) In vitro model systems to study nutraceutical properties. [Tesi di dottorato]

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Abstract

The use of animals for scientific experimentation represents one of the most controversial problems of the human-animal relationship. The ethical aspects of the use of animals in experiments were defined by the principles of the 3Rs: Replacement, Reduction, and Refinement, proposed by William M.S. Russel and Rex L. Burch in 1959. The development and validation of new in vitro cell systems could significantly contribute to the good experimental practice proposed by the “3R” model. The aim of this Ph.D. project was the development of in vitro cellular systems to evaluate the properties of nutritional and nutraceutical compounds such as bioavailability, mechanisms of action, efficacy, and safety. In this work, a comparative study was performed to evaluate the bioaccessibility and bioavailability of vitamins from natural sources and vitamins of chemical synthetic origin. Although the synthetic vitamins and vitamins from natural sources are chemically identical, it is known that the matrix and/or food sources can affect the absorption and bioavailability of a nutrient. Models and assays were selected following EFSA 2018 guidelines. The studies were conducted on different samples: Vitamin C or D of natural origin, vitamin C or D of natural origin in their commercial dosage forms, vitamin C or D of synthetic origin, and vitamin C or D of synthetic origin in their commercial dosage forms. The study design consisted of the setup and validation of the UHPLC-MS methods suitable for the quantification of vitamins C and D in the different samples. The evaluation of bioaccessibility was determined with in vitro simulated digestion protocol. All digested samples were analyzed through UHPLC-MS methods to quantify vitamin C and vitamin D and evaluate their stability to the digestion process. As far as the evaluation of bioavailability was concerned, to estimate the permeability coefficients, PAMPA assay was performed for all samples. Subsequently, in vitro Transwell® assay on Caco-2 cell line was performed and all samples (vitamin C and D) were collected and analyzed through the validated UHPLC-MS method. The results obtained from the analysis of vitamin C showed that after oro-gastro-duodenal synthetic vitamin C undergoes greater degradation as compared to natural vitamin C. As far as vitamin D is concerned, no significant differences were found between vitamin D of natural origin and vitamin D of synthesis. The results obtained from the PAMPA assay suggested that only synthetic vitamin C at high concentration (500 mg/mL) crossed the phospholipid membrane. For vitamin D, the results showed that only sample 2 corresponding to natural vitamin D in dosage form, was absorbed by passive diffusion in the used model system, to indicate that the presence of the phytocomplex and the excipients could help the crossing of the membrane. The results obtained from the analysis of the acceptor compartment of the Transwell® system did not allow to differentiate the absorption of vitamin C and D of natural origin from those of synthetic origin. The cell uptake analysis showed that both natural and synthetic vitamin C are internalized in Caco-2 cells, with a similar percentage of 1.4 and 2.4%. For vitamin D, the cellular uptake did not allow us to differentiate the vitamin D of natural origin from the synthetic one. Other experiments conducted at lower concentrations confirmed this result. The results of the assay based on Transwell® system using Caco-2 cells, did not allow to differentiate the absorption of vitamin C and D of natural origin, from that of vitamin C and D of synthetic origin. The results of the cell uptake assay, performed with the same Caco-2 cell line used for the Transwell® assay, showed that both natural vitamin C and synthetic vitamin C were internalized in the cell and therefore cross the cell membrane. In the last year of PhD research project, a novel organ on chip MIVO® by React4Life model was used to culture human intestinal epithelial Caco-2 cells on a porous membrane under dynamic conditions (i.e. flow) and compared its characteristics to those of the same cells grown in a conventional static in vitro model. Moreover, we studied the vitamin C bioavailability with static (Transwell®) and dynamic (MIVO® Platform) in vitro model systems using Caco-2 cell line. MIVO® platform is based on transparent cell culture chambers with a shape and size similar to the wells of classic 24 well plates. The chambers were connected to a peristaltic pump to ensure a dynamic tissue environment. Firstly, the barrier integrity of Caco-2 monolayer was evaluated in MIVO® platform and Transwell® for 7, 14 and 21 days. Cell barrier integrity was evaluated measuring transepithelial electrical resistance (TEER), luciferin traslocation and ZO-1 expression for tight-junction. The results obtained suggested that TEER value and expressions of tight junction were similar for static and dynamic conditions. However, the permeability of yellow lucifer decreased for the Caco-2 monolayer grown in MIVO® platform grown for 14 days reaching permeability values less than 5%, which represent the reference value accepted for the permeability of paracellular compounds in an intact barrier. To study the applicability of MIVO® model system using Caco-2 human intestinal epithelial cells, the permeability of vitamin C has been performed and the observed effects were compared to those obtained using a conventional static in vitro model. Caco-2 cells cultured under flow for 14 days formed confluent and polarized monolayers that were visually comparable to cells cultured for 21 days under static conditions in Transwell® wells. The data obtained from the permeability assay of natural vitamin C (Rosa canina extract) and synthetic vitamin C suggest no difference between the different sources of vitamin C. In conclusion, cells subjected to dynamic culture conditions were affected by the flow in agreement with Literature data.

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