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Characterizing the biological behavior of Fe3O4 nanoparticles conjugated with acridine orange using in vitro co-culture systems relevant to skin, lung and gut barrier models
1 , 1 , 2 , 3 , 2, 4, 5 , * 1
1  Departament of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania
2  Department of Science and Engineering of Oxide Materials and Nanomaterials, Politehnica University of Bucharest, 011061 Bucharest, Romania
3  Department of Organic Chemistry, Politehnica University of Bucharest, 011061 Bucharest, Romania
4  Research Institute of the University of Bucharest—ICUB, University of Bucharest, 050657 Bucharest, Romania
5  Academy of Romanian Scientists, Ilfov No. 3, 050044 Bucharest, Romania
Academic Editor: Gianrico Spagnuolo


Fe3O4 nanoparticles (NPs) can be conjugated with acridine orange to create a hybrid nanomaterial with unique properties, such as the magnetic characteristics of magnetite and the fluorescence of acridine orange, making them useful for a variety of applications, including cell imaging, drug delivery, and magnetic separation. In this context, we aimed to provide a biological evaluation of this type of NPs using in vitro co-culture models of human skin, lung and intestine. Fe3O4 NPs were obtained by the co-precipitation method from Fe2+ and Fe3+ (1:2 molar ratio). The concentration of acridine orange in an aqueous NH4OH solution was 0.00025%. The product was washed several times with ultrapure water, redispersed, and centrifuged thrice at 6000 × g for 10 minutes. Each supernatant was collected, obtaining three different suspensions of NPs. We developed one model of skin barrier using a co-culture of human keratinocytes (HaCaT cell line) and dermal fibroblasts (CCD-1070Sk cell line), one model of pulmonary barrier made of A549 epithelial cells and MRC-5 fibroblasts, and one model of intestinal barrier composed of human Caco-2 enterocytes and HT-29-MTX mucus-producing intestinal cells. Our results showed that none of the NP suspensions influenced the cell viability of the co-culture systems, suggesting their good biocompatibility on short-term exposure (24 hours) according to the cytotoxicity assays performed. In addition, we observed a specific apical-basal cell polarization in the co-culture systems, being maintained after one-day exposure to the three suspensions of NPs. To sum up, Fe3O4 NPs conjugated with acridine orange could be promising hybrid nanomaterial with good biocompatibility and special properties for future applications in biomedicine.

Keywords: skin; lung; intestine; co-culture; epithelial barriers; iron oxide nanoparticles
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