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. Nanotechnology & skin penetration eg Physical sunscreens (TiO2, ZnO) Dermal risk issues l Exposure l Absorption l Intrinsic Toxicity l TiO2 or ZnO nanoparticles s are colorless water-resistant & insoluble materials s are non-toxic l Zn = essential element (DNA polymerases, DNA stability) 10 0 m10 -1 m10 -2 m10 -3 m10 -4 m10 -5 m10 -6 m10 -7 m10 -8 m10 -9 m10 -10 m (1 m)(1 mm)(1 µm)(1 nm)(100 nm) Salicylic acid (0.5 nm) ZnO nanoparticles (20-50 nm) Water (0.3 nm) (0.1 nm)(10 nm) Hair (80  m) RBC (7  m) Penetrate skin Apparent cut off MW=500 0.9 nm Rhinovirus (25 nm)

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

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

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 Treatment Total absorbed less than 0.03% of product applied as 10  l/cm 2 Receptor phase penetration of Zinc through human epidermal membrane (ug/cm 2 ) over 24hrs. Mean ±SE, n=8 (formulation treatments), n=3 (placebo base and untreated control). 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 Note Gamer et al (2006) pig skin recoveries of 0.8-1.4% of dose

7. Titanium dioxide & Zinc oxide in a topical sunscreen Dussert et al Int J Cosm Sci 19: 119-129 (1997) 100  m TiO 2 ZnO

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 l 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) l Polystyrene NPs, 20 and 200 nm to pig skin (Alvarez- Roman et al., 2004) s No penetration into epidermis / dermis s Accumulation in follicle orifice s But no penetration from follicle l 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. 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 10µm 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!!! Tinkle Environ Health Perspect 2003 But fate also depends on what you do to the skin?

12. Particles 1  m ControlFlexed 60min After 20 strips Surface 0 strips Confirmation by tape stripping & sem 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. What do we predict ? - Desquamation matters! Consider the Molecular Volume Dependence of the Epidermal Exposure Concentration C ss of Soluble Compounds following Stratum Corneum penetration to predict likely penetration of insoluble particles Levels for 30nm particle  10 -18 nmol/mL Adapted from Magnusson et al., J. Invest. Dermatol. 122:993, 2004 In vitro Epidermal Clearance (assume zero) Steady-state Epidermal Exposure Concentration = Maximum flux of compound Desquamation Clearance Return Permeation ++ log k p  0.4482 – 1.729 log MV + 0.4672 log K oct log J max  3.978 – 5.282 log MV C ss = J max k d + k p k d  14-day turnover desquamation rate Levels for MW 800  10 nmol/mL Ratio of levels  10 -19 (!!!)

14. Conclusion of exposure & absorption l The available data on ZnO & TiO 2 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 l 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.