Laser GeneratedPure Nanoparticulate Reference Material

for Risk Assessment Studies

S. Barcikowski, J. Jakobi, A. Hahn, J. Walter, S. Petersen

NanoMed 2009, March 6th, Berlin

JRG Nanoparticles

"Healthy" organic nanoparticles… …from Hannover brewery

0 100 200 300 400 500 600 700 800 900 10000

1

2

3

4

5

6Lindener Spezial Bier

Particle Diameterd

90 = 699.50 nm

d50

= 337.00 nmNumber of Particles: 452

Log-Nomal Fit of Hydrodyn. Diam. Distrib.

= 0.55733xc = 338.801 ±64.378w = 0.688 ±0.199

Rel

ativ

e P

artic

le N

umbe

r F

req

uenc

y P

N (

%)

Hydrodynamic Diameter dh (nm)

Example of a Risk Assessment Approach: Airborne Nanoparticles in the Human Respiratory Tract

Animal Model Human Respiratory Tract

In vitro

Inhalation

Instillation

Aerosol

Colloid

Biological response is correlated to nanoparticle SURFACE

ZrO2: Y

Ref.: Stoeger et al.,GSF Annul. Rep. (2004) 43-48

"Gold Colloid" (Material Data Sheet)

NaN3

Uptake Mediated by Cell Penetrating Peptides

Yang et al., Transferrin-Mediated Gold Nanoparticle Cellular Uptake. Bioconjugate Chem., Vol. 16, No. 3, 2005 495

Transferrin-Mediated Cellular Uptake by Endocytosis. 5-h treatment, green labeling, exc. 488 nm, emiss. 515 nm (A) Control cells (human nasopharyngeal carcinoma cells)(B) cells treated with Au nanoparticles(C) cells incubated with Au-Transferrin nanoparticles;(D) cells treated with Au-albumin nanoparticles; (E and F) cells co-treated with different proportions of Au-TF versus holo-TF (1:2 and 1:5, respectively).

Transferrin Ligand:

not toxic but prolongs

nanoparticle penetration

into cytoplasma

Requirements to Nanoparticulate Reference Material for Risk Assessment Studies

1. Same material composition of aerosol and colloid

- aerosol: inhalation (in vivo)

- colloid: instillation (in vivo / in vitro)

2. High purity

minimized cross effects

3. Size control

Nanoparticle Generation Processes

Form-in-Place

Wet Chemistry

Mechanical Synthesis

Gas Phase Synthesis

• Lithography • Chemical Vapour Deposition • Physical Vapour Deposition

• Sol-Gel-Process• Precipitation of Salts

• Ball Mills, Planet Mills• Kryo-Milling• Homogenisation (organics)

• Flame Hydrolysis• Flame Pyrolysis • Cemical Vapour Synthesis• CO2 Laser Pyrolysis

Start

Ref.: Fonds der Chem. Industrie, Nanobox (2006)

Gas Phase Synthesis / Wet Chemistry

Sol-Gel / Precipitation

- scalability, monodisperse colloids

- chemical precursors and additives

Gasphasensynthese Gasphasenreaktor

Nukleation Wachstum Aggregation

Vorläufer-Dampf

Oxid-Dampf Kügelchen Verklumpung

Reaktions-bereich/Flammzone

Keimbildung

Temperatur / Zeit

Gasphasensynthese Gasphasenreaktor

Nukleation Wachstum Aggregation

Vorläufer-Dampf

Oxid-Dampf Kügelchen Verklumpung

Reaktions-bereich/Flammzone

Keimbildung

Temperatur / Zeit

Gas phase Synthesis- multi-ton scale, gaseous precursors- powders / agglomerates

Precursor Nucleation Aggregation

Nanoparticle Generation Processes

• Availability of precursors Limited Nanomaterials• Agglomeration (powder) Re-Dispersion • Additives and chemicals Purification

Limitations in Risk Assessment Studies

Form-in-Place

Wet Chemistry

Mechanical Synthesis

Gas Phase Synthesis

• Lithography • Chemical Vapour Deposition • Physical Vapour Deposition

• Sol-Gel-Process• Precipitation of Salts

• Ball Mills, Planet Mills• Kryo-Milling• Homogenisation (organics)

• Flame Hydrolysis• Flame Pyrolysis • Cemical Vapour Synthesis• CO2 Laser Pyrolysis

Start

Laser generated

Aerosols

Kammer

Strahlteiler

P

Linse

SampleHolder P

Laser

SampleV

CCD

ELPI

Lens

Beam Splitter

Chamber

Continuous Generation of Nanoparticles in Gasesby Laser Ablation from Solid Targets

Sample Holder

SampleWindow

Inlet

Exit

Experimental Setup Process Chamber

• continuous ablation of any solid target material(e.g. titanium, silver, alloys, …)

• no chemical precursors

Particle Size Distribution During fs-Laser Ablation of Graphite

0%

20%

40%

60%

80%

100%

0.00

70.

030.

06 0.1

0.16

0.25

0.39

0.63

0.98

1.59

2.43

3.93

Aerodyn. Diameter [µm]

Nu

mb

er

Fre

qu

en

cy

Pulse Energy: 50 µJ

Pulse Energy: 300 µJ

S. Barcikowski,et al., ICALEO 2005

Influence of the Process Gas on the Particle Size during fs-laser ablation of Titanium

0%

20%

40%

60%

80%

100%

Press. Air Helium Nitrogen

3.93 µm

2.43 µm

1.59 µm

0.98 µm

0.63 µm

0.39 µm

0.25 µm

0.16 µm

0.10 µm

0.06 µm

0.03 µm

0.007 µm

Nano

Mic

ron a

nd S

ubm

icro

n

S. Barcikowski, A. Hahn, B. Chichkov. J. Laser Appl., Vol. 19, No. 2, May 2007

Particle size distribution during laser ablation of zirconia using different types of laser

0,01 0,1 10

10

20

30

40

50

60

70

80

rel.

fre

qu

en

cy o

f pa

rtic

le n

um

be

r [%

]

aerodynamic particle diameter [µm]

Nd:YAG-Laser: 1 kJ/m CO2-Laser: 20 kJ/m Ti:Sa-Laser: 3 kJ/m

Zirconia

S. Barcikowski, A. Hahn, B. Chichkov, J. Laser Appl., Vol. 19, No.2, pp. 65-73 (2007))

Composition of the nanoparticulate matter

Zr

YO

Zr

Y

Y2O3-doped ZrO2

Laboratory of Biomaterials Dept. of Neurosciences, University of Modena and Reggio Emilia, Italy

S. Barcikowski, J. Walter, A. Hahn, J. Koch, H. Haloui, T. Herrmann, A. Gatti. Proc. LPM 2008. Subm. to Journal of Laser Micro/Nanoengineering (2009)

Composition of the nanoparticles:Energy Electron Loss Spectroscopy

Laboratory of Biomaterials Dept. of Neurosciences, University of Modena and Reggio Emilia, Italy

ZrO2: Y

S. Barcikowski, J. Walter, A. Hahn, J. Koch, H. Haloui, T. Herrmann, A. Gatti. Proc. LPM 2008. Subm. to Journal of Laser Micro/Nanoengineering (2009)

Cr

Fe

Ni

FeCrNi alloy (stainless steel)

Composition of the nanoparticulate matter sampled at the workplace

Laboratory of Biomaterials Dept. of Neurosciences, University of Modena and Reggio Emilia, Italy

S. Barcikowski, J. Walter, A. Hahn, J. Koch, H. Haloui, T. Herrmann, A. Gatti. Proc. LPM 2008. Subm. to Journal of Laser Micro/Nanoengineering (2009)

Start

Laser generated

Colloids

100% pure

fully dispersed and stable

Laser Ablation in Liquids

Pulsed Laser Beam

Liquid

Solid

Pulsed Laser BeamPulsed Laser Beam

Liquid

Solid

safe

unlimited materials and liquids

Laser Generated Colloidal Nanoparticles

300 nm500 nm

50µm

Platinum Silver

500 nm

Copper

Gold ConjugatesNiFe

100 nm

SilicaPVP

FeOx

100 nm

SilicaPVP

FeOx

Iron Oxide

Limitations in fabricating Colloidal nanoparticle alloy from Sm2Co17

Problem: Disproportionation!

Enrichment of

- Co in small

- Sm in big

Nanoparticles.

Possible reason:

Difference (130%) Heat of Evap.

- Co: 377 kJ/mol

- Sm: 166 kJ/mol

cause segregation in

fast (Sm) and slow (Co)

component in laser plume.

PtIr Alloy Nanoparticle Colloid

Pt-Ir Alloy Similar (18%) Heat of Evap.

- Pt: 510 kJ/mol

- Ir: 604 kJ/mol

Electro-Deposition at Neural Electrodes / Implant Surface

50µm 500nm50µm

Uncoated NiTi-Microstructure… …coated with NiTi-Nanoparticles

Menendez et al, JLMN 2009 // Barcikowski et al.,Biomaterialien 2007

PtIr

NiTi

Barcikowski, Hahn, Guggenheim, Reimers, Vogt; unveröffentlicht

Human adipose-tissue derived mesenchymal stem cells grown on NiTi-Nanoparticles

(FE-ESEM at 99.7% humidity)

Start

Stabilisation

Ex-situ Stablisation with Albumin

Albumin ist contained in...

- Blood, Human Serum

- Cell culture media (DMEM, RPMI)

V. Amendola, M. Meneghetti. J. Mater. Chem., 2007, 17, 4705–4710

No Albumin

Stability in 0.07 M KCl

Albumin

U=0 V Brownian

Motion

v

vi

Gold Electrodes

Laser Scattering

Nanoparticle MotionSet-Up

Kinetics

Motion and kinetics of laser-generated Nanoparticles

Vector field of electro-mobility

at U=20 V

(-)

(+)

v

vi

Gold Electrodes

Laser Scattering

Nanoparticle MotionSet-Up

Motion and kinetics of laser-generated Nanoparticles

Kinetics

In-situ Functionalisation

AuS

S

S

S

S S

S

S

S

fs laser beam

variable target (e.g. Au)

variable

ligand

SH COOHNH2

Laser generated NiTi-alloy Nanoparticles

-60

-50

-40

-30

-20

-10

0

10

0.01 0.1 1 10 100

Concentration [mmol/l]

Zet

a P

ote

nti

al [

mV

]

Cysteine

Citrate

In-situ conjugation with citrate or cysteine

Stability in Saline Solutions

Stability of conjugates is evidenced in saline solutions

0.0 0.5 1.0 1.5 2.00.0

0.2

0.4

0.6

conjugated particles pure particles

A (

80

0 n

m/ 3

80

nm

)

[NaCl] [M]

physiological

salinity

400 600 8000.0

0.2

0.4

0.2

0.4 Au NPA

bso

rba

nce

without salt +0.15M NaCl (=physiological) +2M NaCl transfection buffer

Conjugates

Wavelength

0

-20

-40

surf

ace

po

ten

tial [

mV

]

Au NP Conjugates

Stability in Saline Solutions or Buffers

S. Petersen, S. Barcikowski, Advanced Funct. Materials, in press (2009)

Gold Nanoparticles

Nano-Bioconjugates

0 50 100

AUZ

200 nm

200 nm

10 nm

Laser generated Nano-Bio-Conjugates are monodisperse

TEM Micrographs Size distribution

Frequency

Diameter

S. Petersen, S. Barcikowski, Advanced Funct. Materials, in press (2009)

10 100

DLS

0 25 50

TEM

Conclusion

Laser ablation in gases and liquids:

Contribution to systematic studies

on adverse health effects

of engineered nanoparticles?

Nanoparticle reference material:

- purity,

- size,

- composition

matters!

Contributions at LZH-Nanomaterials Group:

Niko BärschAnne HahnJurij Jakobi Ana MenendezChristin MennekingSvea Petersen Laszlo SajtiRamin SattariAndreas SchwenkePhilipp WagenerJohanna WalterJürgen Walter

Funding:European Commission: integrated project LAUNCH-MICRO (NMP2-CT-2005-011795)

German Research Foundation (DFG):Junior Research Group 'Nanoparticles'within Excellence Cluster REBIRTH

Contribution from Univ. Bologna:

Antionetta Gatti

Acknowledgement

Acknowledgement

Contributions at LZH-Nanomaterials Group:Stephan BarcikowskiNiko BärschAnne HahnJurij Jakobi Ana MenendezChristin MennekingSvea Petersen Laszlo SajtiRamin SattariAndreas SchwenkePhilipp WagenerJohanna WalterJürgen Walter

Contribution from Univ. Bologna:

Antionetta Gatti

Thank you !

2 min

1 min

30 s0 s

Duraion of Irradiatin

Laser fragmentation of gold nanoparticles in water

Abs

orpt

ion

[-]

Wavelenghth [nm]