Development of in vitro toxicity test method for safety evaluation of nanoparticles in sunscreen products

摘要

The rapid expansion of nanotechnology has led to a variety of nanoparticles and nanomaterial products with novel physicochemical characteristics. Sunscreen application benefits largely from nanoscales of zinc oxide (ZnO) and titanium dioxide (TiO2) but toxicological profiles of these nanomaterial products are still poorly characterised. This research explores the potential of in vitro methods for toxicity assessment of ZnO and TiO2 sunscreen products. A tiered approach for toxicity testing of sunscreen particles was developed using in vitro assays and skin penetration models. Cytotoxicity was assessed using human skin fibroblasts and A549 lung cells and a range of bioassays (MTS, NRU, ATP and LDH). Physicochemical characteristics of test particles were investigated using analytical techniques. The skin penetration of customised and commercial sunscreens was investigated on freshly excised human abdominal skin using a Franz cell diffusion apparatus followed by three staining techniques (Hematoxylin and Eosin, Gomori trichrome and van Gieson). The results demonstrated that ZnO particles were more toxic than TiO2 particles with regards to altering mitochondrial activities, damaging cell membranes and causing cell death. ZnO nanoparticles (IC50 = 6.64 ± 0.37 ppm) were found to be more toxic than ZnO microparticles (IC50 = 24.66 ± 2.56 ppm). Zn ions were not significantly responsible for cell viability reduction indicating that cytotoxicity was mainly due to particulates rather than released ions. The ATP assay was the most sensitive bioassay selected. SEM/TEM and other techniques revealed that the smaller hydrodynamic size of ZnO nanoparticles could potentially relate to the higher toxicity when compared to microparticle diameters. Although both sunscreen particles played an important role in UVB protection, photomicrographs of human skin suggested that ZnO and TiO2 nanoparticles penetrated through the epidermis following topical application. The significant keratinocyte solubilisation observed was also an indication of adverse effects. Therefore, the use of nanomaterials in sunscreens needs to be regulated and toxicity of nano-products should be evaluated as a very early stage of product development using appropriate test methods. In vitro methods developed in this thesis coupled with the Franz cell diffusion technique could potentially be implemented for toxicity screening strategies of nanoparticles with cosmetic applications.