(422f) Investigations of the Oral Uptake of Titanium Dioxide Nanoparticles Via the Buccal Mucosa

The development of engineered nanomaterials (ENM) and their commercialization for application in (consumer) products, medical and diagnostic devices and/or pharmaceutical drug delivery vehicles presents an enormous challenge for the scientific, regulatory, industrial and public field. Titanium dioxide (TiO₂) nanoparticles, for example, are manufactured worldwide in large quantities and TiO₂ pigments (< 2.5 µm) account for 70% of the total production volume [1]. However, decrease of particle size (from the microscale to the nanoscale) has been identified as a key parameter for an increased toxicity behavior. Regarding the respiratory tract it has been clearly demonstrated that TiO₂, which is one of the priority materials (concerning risk assessment) of the Organisation for Economic Co-operation, causes respiratory tract cancer [2]. However, studies dealing with the oro-gastrointestinal route are still lacking and the basic understanding of how nanoparticles interact with oral biological tissues is still missing. Previously, we demonstrated that nanoparticles are small enough to overcome the barriers of the oral cavity, likely entering systemic circulation and hence, translocate in the human body [3,4]. Thus, in this study we aimed at investigating interactions of three distinct TiO₂ nanoparticles (NM 100, pigment, anatase; NM 101, 7 nm, anatase; NM 105, 22 nm, anatase/rutile) with the oral cavity, in particular the buccal mucosa (lining the cheek area). To correlate particles´ properties to their biological interactions, characterization was carried out with photon correlation spectroscopy (PCS) in physiological media and with electron microscopy. Furthermore, the surface hydrophobicity was quantified with the Rose Bengal adsorption method. Ex-vivo penetration/permeation studies were investigated with static diffusion cells using porcine buccal mucosa. The penetration depth of TiO₂ nanoparticles was visualized by multiphoton microscopy (MPM). Furthermore, intracellular localization was examined via transmission electron microscopy (TEM) and the detected particles were verified with energy filtered TEM (EFTEM) combined with electron energy loss spectroscopy (EELS). Additionally, potential cytotoxicity effects were evaluated in-vitro with human oral TR146 cells and production of reactive oxygen species (ROS) was studied.

The data revealed that the investigated TiO₂ nanoparticles were hydrophilic and exhibited a high aggregation tendency in aqueous and physiological media. The particles penetrated/permeated the buccal epithelium in a size-dependent manner: NM 100 (two primary size distributions: 148 nm and 34 nm) and NM 105 (primary particle size: 22 nm) permeated the upper parts and penetrated into the deepest parts of the mucosa (i.e., basal lamina and connective tissue). However, NM 101 (7 nm particles) only penetrated the upper epithelium. Regarding intracellular localization, most of the particles were found freely distributed in the cytoplasm and not in endocytotic vesicles. These findings suggest that further entering mechanisms, apart from endocytosis, exist in the buccal mucosa. The cell viability/integrity was not compromised and no acute toxicity was observed after 4 and 24 h exposure time. However, NM 105 caused a significant increased metabolic activity (P<0.001) after 4 h, indicating a stress situation of cells. This was due to the production of reactive oxygen species (ROS) after short-time incubation.

It can be concluded that all investigated TiO₂ nanoparticles are able to rapidly penetrate the buccal mucosa. However, the penetration depth is mainly dependent on the particle size. Larger particles (i.e., NM 100 and NM 105) penetrate the upper and the lower parts of the buccal mucosa, suggesting particles´ distribution in the systemic blood circulation and furthermore, translocation in different organs. Smaller particles (i.e., NM 101) fail to penetrate lower parts and are only detected in the superficial layer of the oral epithelium. However, additional studies are necessary to evaluate possible interactions of freely distributed particles (in the cytoplasm) with intracellular proteins, organelles and cell nuclei. Additionally, ROS production of NM 105 may result in inflammation, mitochondria-mediated cell death and/or DNA damage. These data strongly emphasize that the oral cavity should be considered in further risk assessment, since it acts as the first line in defense, not only for oral but also for inhaled nanoparticles.

[1] Baan R., Straif K., Grosse Y., Secretan B., Ghissassi F., Cogliano V., Carcinogenicity of Carbon Black, Titanium Dioxide and Talc, Lancet Oncol 2006; 7: 295-296

[2] Trouiller B., Reliene R., Westbrook A., Solaimani P., Schiestl R., Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic Instability in vivo in Mice, Cancer Res 2009; 69: (22) 8784-878   

[3] Roblegg E., Fröhlich E., Samberger C., Zaversky M., Teubl B., Zimmer A., Evaluation of a Physiological In-Vitro System to Study the Transport of Nanoparticles through the Buccal Mucosa, Nanotoxicology 2012; 6: 399-413.

[4] Teubl B., Meindl C., Eitzelmayr A., Zimmer A., Fröhlich E., Roblegg E., In-vitro Permeability Studies of Neutral Polystyrene Particles Through the Buccal Mucosa, Small, 2013; 9(3): 457-66. doi: 10.1002/smll.201201789