Generation of radicals by doped TiO2 nanopowders in presence of visible and UV light and skin

protection from UV radiation by nanoparticles

A.P. Popov1,2, A. Sarkar3, K. Kordas3,4, M. Meinke5, J. Lademann5, A.V. Priezzhev2,6, R. Myllylä1, V.V. Tuchin1,7,8, J.-P. Mikkola3,

M. Darvin5

1Optoelectronics and Measurement Techniques Laboratory, University of Oulu, Oulu, Finland

2International Laser Center, Moscow State University, Moscow, Rissia 3Technical Chemistry, Department of Chemistry, Umeå University, Umeå,

Sweden 4Microelectronics and Materials Physics Laboratories, Department of Electrical

and Information Engineering, University of Oulu, Oulu, Finland 5Center of Experimental and Applied Cutaneous Physiology, Department of

Dermatology, Universitätsmedizin Charité Berlin, Berlin, Germany 6Physics Department, Moscow State University, Moscow, Rissia

7Institute of Optics and Biophotonics, Saratov State University, Saratov, Russia 8Institute of Precise Mechanics and Control of RAS, Saratov, Russia

2

Outline

• Finland, Oulu, University of Oulu

• TiO2-nano, phototoxicity

• TiO2-nano in skin

• UV protection by TiO2-nano: simulations

• Conclusion

3

Facts about Finland

Independent: 1917European Union: 1995Euro: 1999

Population: 5.3 millionMarked area: 63% of populationHelsinki & beyond: 1 millionOfficial languages: Finnish, SwedishNeighbours: Russia, Sweden, Norway

HELSINKI

OULU

ROVANIEMI

4

Facts about Oulu

Founded: 1605 by King Karl IX of SwedenPopulation: 130.000 (No. 6 in Finland),180.000 (from 2013, No. 5 in Finland)Location: by Gulf of Bothnia Helsinki - 650 km, Arctic Circle – 200 km

5

FACULTIES

FACULTY OF HUMANITIES

FACULTY OF EDUCATION

FACULTY OF ECONOMICS AND BUSINESS

ADMINISTRATION

FACULTY OF SCIENCE

FACULTY OF MEDICINE

FACULTY OF TECHNOLOGY

BOARD RECTOR VICERECTORS

ADMINISTRATION

FOCUS AREAS

BIOCENTER OULU

LABORATORIES

INFOTECH OULU

THULE INSTITUTE

MATHEMATICS

ELECTRONICS

OPTOELECTRONICS AND MEASUREMENTS

MICROEL. & MATERIALS PHYSICS

INFORMATION PROCESSING

COMPUTER ENGINEERING

DEPARTMENTS

Architecture

Mechanical Engineering

Process and Environmental Engineering

Electrical Engineering

Industrial Engineering

TELECOMMUNICATION AND CWC

MICRO AND

NANOTECHNOLOG

Y CENTER

University of Oulu

Founded: 1958Students: 16.000 (No. 3 in Finland)Staff: 3000

6

Optoelectronics and Measurement Techniques Laboratory

Head: Prof. Risto MyllyläFinland Distinguished Professors (FiDiPro):• Valery Tuchin (Saratov State University, Russia), biophotonics• Ghassan Jabbour (King Abdullah University of Science and Technology, Saudi Arabia), printable electronicsStaff (researchers, teachers, students): 50

ResearchAreas: biophotonics (blood, skin), printable electronics (OLEDs, OPV)Biophotonics techniques: DOCT, OCT, Optical tweezers, PAS, TOF

Collaboration

Australia, Finland, Germany, Japan, Korea, Poland, Russia, Sweden, Taiwan, Saudi Arabia, USA

7

Solar spectrum

Absorption in stratosphereSolar spectrum

UV rangeUVC: 100 – 280 nm (absorbed by ozone layer)UVB: 280 – 315 nmUVA: 315 – 400 nmVisible range: 400-750 nm

Wavelength, um Wavelength, nm

reach Earth surface

8

280 300 320 340 360 380 400

0.000

0.001

0.002

0.003

0.004

0.005

0.006U VB U VA

Ha

rm

fu

l e

ffe

ctiv

en

es

s,

r.u

.

W avelength, nm

UV action spectrum

A.P. Popov at al., J. Phys. D: Appl. Phys. 38, 2564-2570 (2005).

200 400 600 800 1000 1200 1400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8Solar spectrum

Sp

. ir

rad

ian

ce

, W

*m-2

*nm

-1

W avelength, nm

Although sun-protective factors are based on effect of UVB light, UVA and even visible lightproduce free radicals and are to be consideredin case of prolonged exposure to solar radiation.

9

Titanium dioxide (TiO2): crystal forms

RutileAnatase

Courtesy “Millenium Chemicals”

10

EPR setup and samples

EPR setup (1.5 GHz, L-band)

EPR = Electron paramagnetic resonanceThe technique is capable for detection of free radicals

Samples with nanoparticles:1. Punch biopsies from porcine ears 2. Paper discs

11

Spectrum of sun simulator

Spectrum of the solar simulator LS 01104 (LOT-Oriel, Germany)

Optical fiber transmittance spectrum

A liquid-filled optical fiber was used to guide light from the solar simulator to the samples. We used UV-VIS fiber (transmittance: 250-700 nm).Use of an optical filter (400-800 nm) allowed filtering out of UV part of spectrum.Light intensities: 77 mW/cm2 (UV+visible) and 55 mW/cm2 (visible). 12

13

TiO2 nanoparticles: SEM photos

TiO2 treatment 1 g TiO2 was heated in N2 atmosphere (s-025) or in 2% NH3 in N2 atmosphere (s-027) at 600 C, for 4 hours.The temperature was ramped at 20 °C/min so that 600 °C is attained in ~ 30 min.

s-027 TiO2s-025 TiO2

TiO2 nanoparticles: photoactivity

300 400 500 600 700 800

0

20

40

60

80

100U V

Ab

so

rpti

on

, %

W avelength, nm

T iO2 s-025

T iO2 s-027

s-027 (absorption in UV+vis.), 6 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16Time, min.

Am

pli

tud

e,

a.u

.

no light_averaged vis._averaged UV+vis._averaged

s-025 (absorption in UV), 4 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16Time, min.

Am

pli

tud

e,

a.u

.

no light_averaged vis._averaged UV+vis._averaged

Up: Absorption spectra of s-025 and s-027 TiO2 (measured by PerkinElmer Lambda 650 spectrometer equipped with an integrating sphere)

Right: Decrease of detected signal from PCA-marker indicated appearance of short-lived free radicals induced by light irradiation

15

s-028 TiO2s-026 TiO2

TiO2 nanoparticles: SEM photos

TiO2 treatment 1 g TiO2 was heated in N2 atmosphere (s-026) or in 2% NH3 in N2 atmosphere (s-028) at 600 C, for 4 hours.The temperature was ramped at 2 °C/min.

16

TiO2 nanoparticles: photoactivity

Up: Absorption spectra of s-026 and s-028 TiO2 (measured by PerkinElmer Lambda 650 spectrometer equipped with an integrating sphere)

s-026 (absorption in UV), 2 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

plitu

de

, a

.u.

no light_averaged vis._averaged UV+vis._averaged

s-028 (absorption in UV+vis.), 2 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

plitu

de

, a

.u.

no light_averaged vis._averaged UV+vis._averaged

Right: Decrease of detected signal from PCA-marker indicated appearance of short-lived free radicals induced by light irradiation

300 400 500 600 700 800

0

20

40

60

80

100

T iO2 s-026

T iO2 s-028

Ab

so

rpti

on

, %

W avelength, nm

17

ZnO and TiO2 anatase: SEM photos

TiO2 anatase, brand HOMBITAN (Sachtleben, former Kemira)

ZnO (Sigma-Aldrich)

18

300 400 500 600 700 800

0

20

40

60

80

100

Ab

so

rpti

on

, %

W avelength, nm

anatase

ZnO

ZnO and TiO2 anatase: photoactivity

Up: Absorption spectra of commercial anatase TiO2 and ZnO (measured by PerkinElmer Lambda 650 spectrometer equipped with an integrating sphere)

ZnO (absorption in UV), 2 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

pli

tud

e,

a.u

.

no light_averaged vis._averaged UV+vis._averaged

Right: Decrease of detected signal from PCA-marker indicated appearance of short-lived free radicals induced by light irradiationNote: UV+vis. curves are less steep (than in case of treated TiO2) due to localization on paper filter surface, no good penetration.

Anatase TiO2 (absorption in UV), 2 samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

pli

tud

e,

a.u

.

no light_averaged vis._averaged UV+vis._averaged

TiO2 anatase, s-025 and s-027 on pig ear skin in vitro: photoactivity

Anatase (absorption in UV) on pig skin, 2 samples

for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

pli

tud

e,

a.u

.

no light_averaged vis._averaged UV+vis._averaged

s-025 (absorption in UV) on pig skin, 2 samples for

each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

plitu

de

, a

.u.

no light_averaged vis._averaged UV+vis._averaged

s-027 (absorption in UV+vis.) on pig skin, 2

samples for each curve

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16

Time, min.

Am

pli

tud

e, a

.u.

no light_averaged vis._averaged UV+vis._averaged

Existing difference between non-irradiated and irradiated with visible or with visibleand UV light is caused by contribution ofpig skin: it also gerenrates free radicals upon irradiation. Detection of particles effect is hardly possible in this case.

20

Skin structure

An OCT image of human skin in vivo (flexor forearm)

epidermis

Stratum corneumEpidermis

Dermisscale bar: 1 mm

Photograph of human corneocytes on a tape strip obtained by Ar+ laser scanning microscopy (λexcit = 488 nm); image size is 250 um x 250 um.

21

Pressing of the tape by a roller Removing of the adhesive film

Application of the emulsion Homogeneous distribution

J. Lademann at al., J. Biomed. Opt.. 10, 054015 (2005).

Tape stripping technique

22

Microscopy of corneocytes

Olympus BX51

Pure skin

Corneocytes + TiO2 (25 nm)

Corneocytes + TiO2 (400 nm)

Scale: 200 um

Courtesy Microel. and Mater. Phys. Labs (Univ. of Oulu)

23

0

0

Dep

th, u

m

Conc. TiO2 particles, ug/cm2

0

0

20

14

Volume concentration of TiO2:V

M

V

V

V

M

V

VNC

0

0

00

0

A.P. Popov et al., J. Opt. Technol. 73, 208-211 (2006).

In-depth particles distribution (by EDX technique)

TiO2 nanoparticles in horny layer

0 2 4 6 8 10 12 14 16 18 20

0

2

4

6

8

10

12

14

16

d = 100 nm

Co

nc

. T

iO2 p

arti

cle

s,

ug

/cm

2

D epth, um

24A.P. Popov et al., J. Biomed. Opt. 10, 064037 (2005).

Qs = s / ( d2) – scattering efficacy factor

s – scattering cross-sectionQa = a / ( d2) – absorption efficacy factor

a – absorption cross-sectiond – particle diameter

Opt. properties of TiO2

particles(rutile modification),

as an example

Calculations by Mie theory

, нм Re(n) – i·Im(n)

310 3.56 – i 1.720

400 3.13 – i 0.008

500 2.82 - i 0.000

40 60 80 100 120 140 160 180 200

0.00

0.01

0.02

0.03

0.04

= 500 nm

= 400 nm

= 310 nm

[Qa

+Q

s(1

-g)]

/d,

nm

-1

Diameter of TiO2 nanoparticle, nm

25

air

epidermis

Optical parameters for SC without nanoparticles

(adopted from V.V. Tuchin, 1998)

A = s(1)/( s

(1) + sm)

d

CQ

V

Nss

s5.1

)1( - scat. coef. of nanoparticles

d

CQ

V

Naa

a5.1

)1(- abs. coef. of nanoparticles

)()1()()( HGMie pApAp

hybrid phase function

2/32

2

)cos21(

1

4

1)(

gg

gp

HG - SC phase function

smss

)1(- scat. coef.

amaa

)1(- abs. coef.

Model of stratum corneum (SC) with nanoparticles

, nm sm, mm-1am, mm-1

310 240 60

400 200 23

Optical parameters for SC with nanoparticles

Attenuation curves of ZnO, TiO2 and Si nanoparticles

26A.P. Popov et al., JQSRT 112, 1891-1897 (2011).

0 20 40 60 80 100 120 140 160 180 200 220

0.00

0.01

0.02

0.03

0.04

0.05

0.06

= 310 nm

[Qa+

Qs*(

1-g

)]/d

, n

m-1

ZnO

Si

T iO2

D iam eter o f partic les, nm

(a)

0 20 40 60 80 100 120 140 160 180 200 220

0.00

0.01

0.02

0.03

0.04

0.05

ZnO

Si

T iO2

= 318 nm

[Qa

+Q

s*(

1-g

)]/d

, n

m-1

D iam eter o f partic les, nm

(b)

0 20 40 60 80 100 120 140 160 180 200 220

0.00

0.01

0.02

0.03

0.04

0.05

T iO2

S i

ZnO

= 360 nm

[Qa

+Q

s*(

1-g

)]/d

, n

m-1

D iam eter o f partic les, nm

(c)

0 20 40 60 80 100 120 140 160 180 200 220

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

= 400 nm

D iam eter o f partic les, nm

(d)

ZnO

TiO2

S i

[Qa+

Qs*(1

-g)]

/d,

nm

-1

Attenuation curves for = 310- (a), 318- (b), 360- (c) and 400-nm (b) radiation vs. particle size, according to the Mie theory; g – scattering anisotropy factor, d – particle diameter.

300 320 340 360 380 400

2

3

4

5

6

7

8

9

10

11

12 T iO2

S i

ZnO

pure skin

Ab

so

rpti

on

, %

W avelength, nm

(a)

300 320 340 360 380 400

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

T iO2

S i

ZnO

pure skin

Re

fle

cta

nc

e,

%

W avelength, nm

(b)

300 320 340 360 380 400

15

20

25

30

35

40

45

50 T iO

2

S i

ZnO

pure skin

Tra

ns

mit

tan

ce

, %

W avelength, nm

(c)

300 320 340 360 380 400

40

50

60

70

80

90

100

110

120

130

140

150

T iO2

S i

ZnO

Dia

me

ter

of

pa

rtic

les

, n

m

W avelength, nm

(d)

Absorption (a), reflectance (b) and transmittance (c) of light through stratum corneum. Calculations were performed for the following optimal sizes of TiO2, Si and ZnO particles, respectively: a) = 310 nm: 62, 55 and 45 nm, b) = 318 nm: 62, 58 and 52 nm, c) = 360 nm: 98, 64 and 92 nm, d) = 400 nm: 122, 70 and 140 nm. Section (d) corresponds to the optimal size-wavelength dependence.

27

Effect of optimal sizes

• Doping of TiO2 nanoparticles in 2% NH3 in N2 atmosphere causesappearance of absorption in visible spectral range.

• Upon irradiation with visible light (55 mW/cm2, 400-700 nm) freeshort-lived radicals formed by TiO2 particles are detected by an L-band (1.5 GHz) EPR spectrometer.

• Effect particles effect is hardly possible if they are localized on pigskin in vitro due to pronounced contribution of skin to radicalproduction.

• In addition, optimal sizes of ZnO, TiO2 and Si nanoparticles arecalculated and their effect on skin protection from UV light isestimated.

Conclusion