The Impact of Titanium Dioxide Nanoparticles on Keratinocyte Proliferation and Differentiation

TiO₂ nanoparticles are still widely used as food additives in the United States for their whitening properties, UV absorption capabilities, and their potential usage as antibacterial reagents. In fact, these particles are considered valid organic additives and can be incorporated at concentrations as high as 1%. When ingested these particles can circulate in the body coming in contact with viable cells and the long term impacts of TiO₂ build up is currently of concern for the European Food Safety Authority [1]. Additionally, even though nanoparticles may not penetrate the stratum corneum and hence do not come into direct contact with viable keratinocytes, if they are used in wound care treatments, they can come into direct contact with viable epidermal and dermal cells and influence cell function and tissue healing. In early experiments producing skin equivalents with keratinocytes exposed to 0.8 mg/mL rutile TiO₂ nanoparticles, we observed nanoparticle aggregation with premature stratum corneum formation and aberrant expression of filaggrin, a late stage multifunctional differentiation marker.<br/>Keratinocytes were grown with lethally irradiated 3T3 cells [2] with media modifications previously described [3]. At 70% confluence feeder cells were removed and cultures were treated with rutile TiO₂ (0.4 mg/mL); no TiO₂ cultures served as control. After 24 hours cultures were rinsed twice with Ca and Mg free phosphate buffered saline, resuspended in buffered saline with BSA and cells sorted at the Stony Brook University Flow Cytometry Core Facility using a FACS Aria IIIU cell sorter.<br/>Cells associated with TiO₂ were separated from TiO₂-free cells based on side scatter using flow cytometry; cells showing low and high side scatter were collected. In these cultures, 90% of the cells exhibited low side scatter, consistent with the absence of nanoparticles. This compares with a low and high scatter population of 57.2% and 42.8%, respectively, in the TiO₂ treated cultures.<br/>The impact of 0.4 mg/mL TiO₂ on keratinocyte proliferative capacity was also explored as a function of colony forming efficiency (CFE), and the colony forming efficiency of keratinocytes with low and high side scatter was determined; TiO₂-free keratinocyte cultures served as control [4]. 12 culture dishes containing 1000 cells each were plated: 4 control plates (low side scatter), 4 plates of TiO₂ treated high side scatter, and 4 plates of TiO₂ treated low side scatter. Following 12 days of incubation, each culture dish was rinsed with Ca and Mg free phosphate buffered saline and stained with Rhodamine B. Colonies ≥ 1 mm were counted. In these cultures, CFEs are about three-fold lower in the high side scatter population suggesting NP inhibition of plating efficiency or proliferation. To assess the full impact of these NPs in this system and in skin equivalents, ongoing work includes SEM analyses to determine NP location within the cell and differentiation assessment by RT-PCR to determine overall skin development.<br/><br/><br/>[1] Flavourings, Younes, M., Aquilina, G., Castle, L., Engel, K., Fowler, P., Fernandez, M. J. F., Fürst, P., Gundert-Remy, U., Gürtler, R., Husøy, T., Manco, M., Mennes, W., Moldeus, P., Passamonti, S., Shah, R., Waalkens Berendsen, I., Wölfle, D., Corsini, E., . . . Wright, M. (2021). EFSA Journal, 19(5). https://doi.org/10.2903/j.efsa.2021.6585<br/>[2] Rheinwald, J. G., & Green, H. (1975). Cell, 6(3), 331–343. https://doi.org/10.1016/s0092-8674(75)80001-8<br/>[3] Randolph, R. K., & Simon, M. (1993). The Journal of biological chemistry, 268(13), 9198–9205.<br/>[4] Barrandon, Y., & Green, H. (1987). Proceedings of the National Academy of Sciences of the United States of America, 84(8), 2302–2306. https://doi.org/10.1073/pnas.84.8.2302