BIOMEDICAL STUDIES ITN Annual Report  2004 147 Nuclear Microscopy Studies of Skin Permeability to Nanoparticles T. Pinheiro, L.C. Alves, A. Verssimo, F. Marujo, R. Silva 1 , J.N. Silva 1 , P. Filipe 1 , M. Amin 2 Objectives Nanoparticles of TiO 2 and ZnO are widely used in commercial sunscreens by their capacity to scatter UV wavelengths of sunlight. Skin exposure to commercial products containing nanoparticles of Ti, Zn, and Si oxides, among others and their trans-epidermal diffusion has been studied using nuclear microscopy techniques. One of the objectives of the work is to assess the percutaneous penetration depth of Ti oxides. Therefore, methodologies were adjusted to enable the validation of elemental distribution maps or profiles with high-resolution images originated in transmission mode (STIM, Scanning Transmission Ion Microscopy). Results Project EC/QLK4-CT-2002-02678 The permeability of mammalian skin (pig and human) to TiO2 particles (<20nm) is being studied. Several emulsions containing micronised Ti were tested in pig skin. As far as human skin is concerned, three commercial products and one of the test emulsions containing micronised Ti were studied. Skin is a stratified tissue and the precise identification of skin layers is needed to ascertain the penetration depth of the physical filter from the formulation. Fig.1. High-resolution transmission image (A) of pig skin evidencing the horny layer (B) and the Ti distribution map below. The overlapping of images (bottom right) show that Ti does not surpass the horny layer of skin. High-resolution STIM images can be used to identify different layers of skin, as cell resolution is achieved. As shown in Fig. 1, different nuclear microscopy images obtained (e.g., X-rays, backscattered particles and transmitted particles) provide information related with the structure of the tissue and elemental distribution. The profiles of P, Cl, and K enable to distinguish major skin strata, which are better evidenced by high- resolution transmission images of skin sections. There are no evidence of penetration of Ti below the horny layer of the skin (Fig.2). Fig. 2. High-resolution transmission image of a human skin section exposed to a product containing Ti and Zn oxides. Ti distribution image (colour) is overimposed. The horny layer (sc), epidermis (epi) and dermis (d) regions are evidenced. Below the elemental profiles of P, Cl, Ti and Zn across skin are plotted. Published, accepted or in press work 1. P. Aguer, et al., Skin morphology and layer identification using different STIM geometries, Nucl Instrum. and Meth., in press. 2. J. Silva, et al., Localisation of titanium dioxide nanoparticles in human skin, J. Europ. Academy of Dermatol. Venereol. 2004;18 Suppl.2:170-171. ________________________ 1 Clnica de Dermatologia, FM-UL, Portugal 2 Universidade do Minho, Portugal d epi sc 0 5000 10000 15000 20000 25000 30000 Concentration (ug/g) P Cl 1 10 100 1000 10000 100000 0 50 100 150 200 250 Pixels Concentration (ug/g) Ti Zn d epi sc 0 5000 10000 15000 20000 25000 30000 Concentration (ug/g) P Cl 1 10 100 1000 10000 100000 0 50 100 150 200 250 Pixels Concentration (ug/g) Ti Zn 10 µm Ti A B min max min max 10 µm 10 µm Ti A B min max min max min max