The latest science (including safety) on nanotechnology and

skin penetration.

The latest science (including safety) on nanotechnology and

skin penetration.

Michael Roberts PhD DScSchool of Medicine

University of QueenslandPrincess Alexandra Hospital

Australia

Nanotechnology & skin penetrationeg Physical sunscreens (TiO2, ZnO) Nanotechnology & skin penetrationeg Physical sunscreens (TiO2, ZnO)

Dermal risk issues Exposure Absorption Intrinsic Toxicity

TiO2 or ZnO nanoparticles TiO2 or ZnO nanoparticles are colorless water-resistant & are colorless water-resistant &

insoluble materialsinsoluble materials are non-toxicare non-toxic

Zn = essential element (DNA Zn = essential element (DNA polymerases, DNA stability)polymerases, DNA stability)

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) (1 nm)(100 nm)

Salicylic acid (0.5 nm)

ZnO nanoparticles (20-50 nm)

Water (0.3 nm)

50nm50nm

(0.1 nm)(10 nm)

Hair (80 m) RBC (7 m)

Penetrate skin

Apparent cut off MW=500

0.9 nm

Rhinovirus (25 nm)

Exposure & absorptionExposure & absorption

In general Nanoparticles preferentially accumulate in

hair follicle openings Stratum corneum penetration limited to upper

layers

Our experiences – zinc oxide nanoparticles applied to human skin

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

50nm

TEM of coated ZnO

Particle Size (nm)

Spectral transmittance in aqueous solution

PCS size distribution Cross et al Skin Physiol Pharmacol, in press

Our experiences – zinc oxide nanoparticlesOur experiences – zinc oxide nanoparticlesConclusion:

• 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

Our experiences – zinc oxide nanoparticlesOur experiences – zinc oxide nanoparticles

Treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 2 3 4

Zn

Ab

sorp

tio

n i

n 2

4hrs

(u

g/c

m2)

ZinClear-60_CCT ZinClear-Commercial Placebo Cream Untreated

Total absorbed less than 0.03% of product applied as 10l/cm2

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).

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

Titanium dioxide & Zinc oxide in a topical sunscreen

Titanium dioxide & Zinc oxide in a topical sunscreen

Dussert et al Int J Cosm Sci 19: 119-129 (1997)

100m

TiO2

ZnO

Titanium dioxide – stratum corneum & follicular levels after stripping in vivo

Titanium dioxide – stratum corneum & follicular levels after stripping in vivo

RELATIVE HORNYLAYER

THICKNESS[%]

0

100

TITANIUM CONCENTRATION [µg/square centimetre tape]

54

14

NUMBER OF

TAPE STRIPPING

1

5

10

15

40

50

60

780,05

0,2

0,4

0,4

0,3

1

2

3

Some absorption into upper layers of stratum corneum, but no penetration

Lademann et al., 1999

Other studiesOther 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)

Quantum dots & pig skin….ButQuantum 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

• 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?

But fate also depends on what you do to the skin?

2 & 4µm remain on surface

0.5µm entry via a tear

1µm & flexing 30 min

1µm & flexing 60 min

Particles1 m

Control Flexed 60min

One micron particle at a depth of twenty tape stripsOne micron particle at a depth of twenty tape strips

After 20 strips

Surface 0 stripsSurface 0 strips

Confirmation by tape stripping & semConfirmation 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

What do we predict? - Desquamation matters!What do we predict? - Desquamation matters!

Molecular Volume (mL/mol)

1e+2 1e+3 1e+4 1e+5 1e+6 1e+7 1e+8

Css

(nm

ol/m

L)

1e+5

1e+0

1e-5experiment in vitrotheory (in vitro)theory (in vitro) with safety factor x100

10nm-radiusnanoparticle

30nm-radiusnanoparticle

IN VITRO

Molecular Volume (mL/mol)

1e+1 1e+2 1e+3 1e+4 1e+5 1e+6 1e+7 1e+8

Css

(nm

ol/m

L)

1e+5

1e+0

1e-5

1e-10

1e-15

1e-20

experiment in vitroin vivo (estimated from in vitro)theory (in vitro)theory (in vivo)theory (in vitro) with safety factor x100theory (in vivo) with safety factor x100

10nm-radiusnanoparticle

30nm-radiusnanoparticle

IN VIVO(desquamation)

IN VITRO

Molecular Volume (mL/mol)

10 100 1000

log

(max

imum

flux

) (

nmol

/cm

2 /h)

-6

-4

-2

0

2

4

6

8

best fit95% confidence

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

Levels for 30nm particle 10-18 nmol/mL

Adapted from Magnusson et al.,

J. Invest. Dermatol.122:993, 2004

In vitro

EpidermalClearance(assume zero)

Steady-stateEpidermalExposure

Concentration

=

Maximum flux of compound

DesquamationClearance

ReturnPermeation

++

log kp 0.4482 – 1.729 log MV + 0.4672 log Koct

log Jmax 3.978 – 5.282 log MV

Css =Jmax

kd + kpkd 14-day turnover desquamation rate

Levels for MW 800 10 nmol/mL

Ratio of levels 10-19 (!!!)

Conclusion of exposure & absorption

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.