The latest science (including safety) on nanotechnology and skin penetration.

1. The latest science (including safety) on nanotechnology and skin penetration. Michael Roberts PhD DSc School of Medicine University of Queensland Princess Alexandra Hospital Australia

2. Dermal risk issues Exposure Absorption Intrinsic Toxicity TiO2 or ZnO nanoparticles are colorless water-resistant & insoluble materials are non-toxic Zn = essential element (DNA polymerases, DNA stability) Nanotechnology & skin penetrationeg Physical sunscreens (TiO2, ZnO) Apparent cut off MW=500 0.9 nm Penetrate skin 100 m 10-1 m 10-2 m 10-3 m 10-4 m 10-5 m 10-6 m 10-7 m 10-8 m 10-9 m 10-10 m (1 m) (1 mm) (1 µm) (100 nm) (1 nm) (10 nm) (0.1 nm) RBC (7 m) Hair (80 m) Rhinovirus (25 nm) ZnO nanoparticles (20-50 nm) Salicylic acid (0.5 nm) Water (0.3 nm)

3. Exposure & absorption • In general • Nanoparticles preferentially accumulate in hair follicle openings • Stratum corneum penetration limited to upper layers

4. Particle Size (nm) 50nm Our experiences – zinc oxide nanoparticles applied to human skin • Epidermal membrane in Franz cells with PBS & DC-30 2% • ZinClear o/w sunscreen & placebo for 24 hr • Zn assayed using ICP-MS after acidifying solution • TEM TEM of coated ZnO Spectral transmittance in aqueous solution Cross et al Skin Physiol Pharmacol, in press PCS size distribution

5. Our experiences – zinc oxide nanoparticles • Conclusion: • Electron micrographs of human skin show ZnO nanoparticle mineral components present on the surface of the skin & around desquaming corneocytes • No penetration into the underlying intact stratum corneum was observed • Multiphoton images also showed zinc oxide & 10nm Cerium Oxide was retained in follicle openings & around desquamating corneocytes • Stretching or flexing the skin did not affect particle distribution –stays on stratum corneum surface Cross et al Skin Physiol Pharmacol, in press

6. Our experiences – zinc oxide nanoparticles Receptor phase penetration of Zinc through human epidermal membrane (ug/cm2) over 24hrs. Mean ±SE, n=8 (formulation treatments), n=3 (placebo base and untreated control). Total absorbed less than 0.03% of product applied as 10l/cm2 Note Gamer et al (2006) pig skin recoveries of 0.8-1.4% of dose Treatment Conclusion: Zinc penetration may be observed but is likely to be negligible and as hydrolysed to zinc rather than as zinc oxide Cross et al Skin Physiol Pharmacol, in press

7. TiO2 100m ZnO Titanium dioxide & Zinc oxide in a topical sunscreen Dussert et al Int J Cosm Sci 19: 119-129 (1997)

8. Titanium dioxide – stratum corneum & follicular levels after stripping in vivo Some absorption into upper layers of stratum corneum, but no penetration Lademann et al., 1999

9. Other studies • Negatively charged fluorescein particles of 50 and 500nm penetrated pig skin whereas 100 & 200 did not nor did neutral & positively charged particles (Kohli & Alpar, 2004)(not found with carboxylated nanoparticles in human skin) • Polystyrene NPs, 20 and 200 nm to pig skin (Alvarez-Roman et al., 2004) • No penetration into epidermis / dermis • Accumulation in follicle orifice • But no penetration from follicle • Nano titanium oxide 10-100nm retained on outermost layer of human skin in vivo after 6 hr with none being detected in deeper SC, epidermis or dermis in punch biopsies (Pflucker et al, 2001; Schulz et al 2002)

10. Quantum dots & pig skin….But • Quantum dots penetrate porcine skin • All 3 coated materials had penetrated by 8 hr • Localisation affected by surface charge • Conclude skin could serve as entry point for a diversity of engineered nanostructures • Pig • pH 8.3 PEG, pH 9.0 COOH • Perfused • Lymph nodes

11. But fate also depends on what you do to the skin? • Red fluorescein iso-thiocyanate (FITC) - conjugated dextran beads to human skin (back) for 30min. • Skin either flexed at 45°, 20 flexes/minute ( with double-sided tape) or left flat, for 15, 30, or 60 min • Flexing time critical for 0.5 & 1 µm beads penetration into epidermis • 15 min – seen in 2 of 11 skin samples (18%) • 30 min - in 5 of 12 samples & • 60min - in 9 of 16 samples; also penetration into the dermis for two samples • No particle penetration observed without flexing at any time or size!!! 10µm Tinkle Environ Health Perspect 2003

12. Confirmation by tape stripping & sem Control Surface 0 strips Flexed 60min Surface 0 strips Particles 1 m After 20 strips • Hydrated 24-48hr • Possible that mechanical force & particle size (100nm-500nm)  skin penetration? Not seen for ZnO 30nm flexed • No diffuse reflectance • No particles in follicle Tinkle Environ Health Perspect 2003

13. Maximum flux of compound Steady-state Epidermal Exposure Concentration = Desquamation Clearance Epidermal Clearance (assume zero) Return Permeation + + log Jmax 3.978 – 5.282 log MV Jmax Css log kp  0.4482 – 1.729 log MV + 0.4672 log Koct = kd+ kp kd  14-day turnover desquamation rate Levels for MW 800 10 nmol/mL Levels for 30nm particle 10-18 nmol/mL Ratio of levels  10-19 (!!!) What do we predict? -Desquamation matters! Consider the Molecular Volume Dependence of the Epidermal Exposure Concentration Css of Soluble Compounds following Stratum Corneum penetration to predict likely penetration of insoluble particles In vitro Adapted from Magnusson et al., J. Invest. Dermatol. 122:993, 2004

14. Conclusion of exposure & absorption • The available data on ZnO & TiO2 suggests it is unlikely that significant amounts will penetrate through human stratum corneum either directly or via the hair follicles & result in any local or systemic toxicity • Conclusion is similar to Australia’s Therapeutic Goods Agency (TGA’s) perspective ” …..The weight of current evidence is that they remain on the surface of the skin & in the outer dead layer (stratum corneum) of the skin.” Acknowledgements: Australian National Health & Medical Research Council (NHMRC), Advanced Nanotechnology, my staff (Dr M Sarkar, Dr O Jepps, Dr S Cross, Dr J Grice), colleagues (Dr A Zyvagin, Dr Y Anissimov) & graduate students & Dr Sally Tinkle for sharing her slides – and to CTFA & NHMRC for inviting me.