Food proteins conjugates to magnetic nanoparticles : evaluation of their cellular toxicity

Introduction Advances in processes for producing nanostructured materials coupled with appropriate formulation strategies has made possible the production and stabilisation of magnetic nanosupports that have potential applications in biochemical and biotechnological fields. This kind of support is generally synthesized by encapsulating magnetic materials with a polymer layer. The interest for magnetic nanosupports is not only limited to the obvious ease of their separation under micro- and nanofluidic conditions, or to their entrapment in coatings and films. Most relevant is the fact that nanostructures can be conjugated to biologically active molecules, including hormones, antibodies, drugs, and various peptides, taken up by cells, and circulated among tissues expressing their cognate receptors (1). For all the studies described above, it is fundamental to set up appropriate tests aimed at assessing the citotoxicity of nanoparticles. In this work we investigated the citotoxicity of unmodified and protein-coniugated dextran-coated nanoparticles using the human intestinal cell lines HT-29 and Caco2, differentiated in vitro toward an enterocityc phenotype. Materials and methods Cytotoxicity was assessed by the colorimetric tetrazolium assay (2) performed in HT-29 cells differentiated in RPMI 1640 medium. The cell proliferation rate was quantified by bromodeoxyuridine (BrdU) incorporation into DNA, while the apoptosis was monitored by quantification of the activity of caspase 3 and 7 (Apo-ONE® assay). Effects on tight junctions permeability were evaluated by TEER measurements in differentiated Caco2 cells. Results: Dextran-coated iron oxide magnetic nanoparticles were modified through simple chemical procedures in order to obtain a functionalized and activated coating which allowed easy covalent binding of different bioactive proteins. Proteins considered in our studies included food allergens, enzymes, and antibodies to food proteins. Immunological approaches were used to demonstrate the coupling of the proteins to the activated dextran-coated nanoparticles. Cell viability after 24-48 hours incubation with unmodified and conjugated nanoparticles showed the absence of any citotoxic effect, indicating a full biocompatibility of these particles with this in vitro intestinal cellular model. Both the cell proliferation rate and the apoptotic marker were unaffected by 24 hours incubation with the same nanoparticles. TEER measurement in differentiated Caco2 cells demonstrated that both unmodified and conjugated nanoparticles increase the Trans-Epithelial Electrical Resistance, thus revealing a direct effect on the paracellular permeability of intestinal cells. This result suggests that both unmodified and conjugated nanoparticles may act as protective agents against foreign molecules that are potentially dangerous for the integrity of the epithelial barrier made by the cellular tight junctions. Further studies will assess the use of the conjugates between magnetic nanoparticles and bioactive proteins as biological tracers in order to monitoring the intracellular and/or intratissutal path of specific proteins, with particular reference to food allergens of protein nature and to food-derived compounds of known toxicity.