Background: There is increasing concern over the local and systemic side effects of TiO2 and ZnO coated nanoparticles widely used in sun blockers. Objective: To determine the localization and possible skin penetration of TiO2 and ZnO nanoparticles, dispersed in 3 sunscreen formulations, under realistic in vivo conditions in normal and altered skin. Methods: Nuclear microscopy techniques provided spatially resolved quantitative analysis of Ti and Zn nanoparticle distributions in transversal cryosections of skin obtained by biopsy with no further treatment. A test hydrophobic formulation containing coated 20-nm TiO2 nanoparticles and 2 commercial sunscreen formulations containing TiO2 alone or in combination with ZnO were tried, taking into account realistic use conditions by consumers and compared with the recommended standard condition for the sun protection factor test. The protocols consisted of an open test. Results: Following a 2-hour exposure period of normal human skin to TiO2- and ZnO-containing sunscreens, detectable amounts of these physical blockers were only present at the skin surface and in the uppermost stratum corneum regions. Layers deeper than the stratum corneum were devoid of TiO2 or exogenous ZnO, even after 48 h of exposure to the sunscreen, under occlusion. Deposition of TiO2 and ZnO nanoparticles in the openings of the pilosebaceous follicles was also observed, suggesting a preferential fixation area. Penetration of nanoparticles into viable skin tissue could not be detected. Conclusions: TiO2 or ZnO nanoparticles are absent or their levels are too low to be tested under the stratum corneum in human viable epidermal layers. Therefore, significant penetration towards the underlying keratinocytes is unlikely.
Nuclear microscopy is a technique based on a focused beam of accelerated particles that has the ability of imaging the morphology of the tissue in vivo and of producing the correspondent elemental maps, whether in major, minor, or trace concentrations. These characteristics constitute a strong advantage in studying the morphology of human skin, its elemental distributions and the permeation mechanisms of chemical compounds. In this study, nuclear microscopy techniques such as scanning transmission ion microscopy and particle induced X-ray emission were applied simultaneously, to cryopreserved human skin samples with the purpose of obtaining high-resolution images of cells and tissue morphology. In addition, quantitative elemental profiling and mapping of phosphorus, calcium, chlorine, and potassium in skin cross-sections were obtained. This procedure accurately distinguishes the epidermal strata and dermis by overlapping in real time the elemental information with density images obtained from the transmitted beam. A validation procedure for elemental distributions in human skin based on differential density of epidermal strata and dermis was established. As demonstrated, this procedure can be used in future studies as a tool for the in vivo examination of trans-epidermal and -dermal delivery of products.
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