Evaluation of Magnetic Nanoparticle Internalization in CaCo-2 Cells by AC Biosusceptometry After Simulated In Vitro Digestion

The low solubility, permeability, and stability of various drugs in the gastrointestinal tract make oral administration a significant challenge. In this context, nanotechnology emerges as a promising strategy, particularly through the use of nanoengineered drug delivery systems. Among the experimental models used to study gastrointestinal absorption, the CaCo-2 cell model stands out as a widely employed tool to simulate the intestinal epithelium. However, combining this model with in vitro digestion simulation offers a more physiologically relevant approach. Furthermore, to monitor the internalization and quantification of magnetic nanoparticles (MNPs), sensitive techniques such as Alternating Current Biosusceptometry (ACB) are required.In this study, we evaluated the internalization and bioavailability of manganese-doped iron oxide MNPs (MnFe₂O₄), citrate-coated, in CaCo-2 cells after simulated in vitro digestion. The cells were cultured in medium supplemented with fetal bovine serum and maintained in an incubator at 37 °C with 5% CO₂. Upon reaching confluency, they were trypsinized, counted, and seeded for subsequent exposure to the digested MNPs. Digestion was performed in three sequential phases (oral, gastric, and intestinal), following the protocol by Minekus et al. (2014). After exposure at different time points, samples were collected and divided into supernatant (non-internalized MNPs) and trypsinized samples (MNPs associated with the cells). Quantification was performed using ACB.The results showed significant variations in internalization profiles over time and among the different digestion phases. The in vitro digestion directly influenced nanoparticle bioavailability, highlighting the impact of the simulated gastrointestinal environment on nanoparticle–cell interactions.