NanoMed 2009, Berlin Nanoparticle-bioconjugates by laser ablation- a novel method aiming at pure...

NanoMed 2009, Berlin


Nanoparticle-bioconjugates by laser ablation- a novel method aiming at

pure drug and gene deliveryS. Petersen, S. Barcikowski

JRG Nanoparticles


Nano Medicine

Imaging

Drug DeliveryTherapy

Targeting

Gao et al. 2004

© MagForce Nanotechnolgies

Nanobiomaterials

Science Photogallery


Imaging

Drug DeliveryTherapy

Targeting

Gao et al. 2004

© MagForce Nanotechnolgies

Nanobiomaterials

Science Photogallery

Core/Shell

Nanoparticle


Functional Nanoparticles

Core : Low toxicity, stable, visualizable, easily conjugable (e.g. gold nanoparticles)

Coating: Tailored biological function

Prerequisite for Nanomedicine

Requirements for Production Method

No side effects, cytotoxicity

Rapid (single step) preparation method

Size control

Material variety

Multifunctionality


Femtosecond laser ablation in liquids

• 100% pure• Material variety

• Fully dispersed and stable• Safe

fs-laser


Electron affinity of laser generated gold nanoparticles

fs-laser

variable target (e.g. Au)

Au+, Au3+

Nanoparticle

= electron acceptor

Ligand = electron donor

Conjugation

SH COOHNH2

Compagnini, G et al, J. Mater. Res. 2004, Vol.19, No.10Meunier group: Journal Am. Chem. Soc. 2004, Vol 126, S. 7176 – 7177

S. Petersen, J. Jakobi, S. Barcikowski. Biomaterialien 8 (3) 2007. 155-156S. Petersen, S. Barcikowski. Appl. Surf. Sc. (2008). in press

Mafune group, J. Phys. Chem. C, 2007, 10.1021Meunier group, J. Phys. Chem. B, 2004, 108, 16864-16869


In-situ bioconjugation

e.g. gold nanoparticles with thiolized single stranded oligonucleotides (23mer)

AuS

S

S

S

S S

S

S

S

fs laser beam

variable target (e.g. Au)

variable reagent (e.g. thiolized

ssoligonucleotide)


1.) Negligible degradation of biomolecule

Gel electrophoresis

Deg (x) = 1- I(x)/ I(0)

Evaluating optimal laser and process parameter

2.) Sufficient nanoparticle productivity

SH


Degrades the biomolecule during laser irradiation? SH

Petersen et al. Adv. Funct. Mater. 2008


Full integrity of biomolecules can be guaranteed

Degrades the biomolecule during laser irradiation? SH



Productivity during in-situ bioconjugation

50 100 150 200-8

-6

-4

-2

0ablated mass after 53 s [µg]

pulse energy [µJ]

foca

l pos

ition

[mm

] 10

20

30

40

Productivity

SH

Petersen et al. APSUSC 2008


Productivity and Purity during in-situ bioconjugation

50 100 150 200-8

-6

-4

-2

0ablated mass after 53 s [µg]

pulse energy [µJ]

foca

l pos

ition

[mm

] 10

20

30

40

Productivity

50 100 150 200-8

-6

-4

-2

0

pulse energy [µJ]fo

cal p

osi

tion [m

m]

80%85%90%95%100%

degree of integrity after 53s

Integrity

100% integrity of DNA at moderate nanoparticle productivity: 1mg/h

SH



300 400 500 600 700 8000,0

0,2

0,4

0,6

0,8

1,0

1,2

Abs

orba

nce

Wavelength [nm]

without ssO 0.4 µM 1.5µM 3µM 5µM 8µM 10µM

ssO concentration260 nm

520 nmGold-DNA-Conjugate

AuSH

SH

AuSHSH

SHSH

Gold

Au

Petersen et al. JLMN 2009

UV-Vis Spectra of BioconjugatesSH


0 50 1000,0

0,1

0,2

0 25 50 10 100

Fre

quen

cy

AUZ

Diameter [nm]

DLS

200 nm

10 nm

200 nm


Size quenching leading to monodisperse bioconjugates SH


Tailored Sizes of Bioconjugates

0,5 1,0 1,5 6 8 10

10

20

30

40d

h50

[nm

]

DNA concentration [µM]

0,0 0,1 0,2 0,3 0,4 250 500 750 1000

Fixed Fluence: 0.3 J*cm-2

SH



0,5 1,0 1,5 6 8 10

10

20

30

40d h

50 [nm

]

DNA concentration [µM]

0,1 0,2 0,3 0,4 250 500 750 1000

0,0 0,1 0,2 0,3 0,4 250 500 750 1000

Fluence [J*cm-2]

Fixed Fluence: 0.3 J*cm-2

Fixed DNA Concentration: 3µM

Tailored Sizes of Bioconjugates

Screening of different sizes in biomedical assays is enabled

SH




In-situ bioconjugation with unmodified oligonucleotides

100 nm

Monodisperse Bioconjugates


4000 3500 3000 2500 2000 1500 1000 500

94,0

94,5

95,0

95,5

96,0

96,5

97,0

97,5

98,0

98,5

99,0

99,5

100,0

100,5

101,0

ggcgactgtgcaagcaga

Tra

nsm

issi

on [%

]

Wavenumber [cm-1]

DNA Reference NP-DNA

Successful biological recognition of DNA (129bp) by PCR

No alteration due to laser irradiation

In-situ bioconjugation with double stranded DNA

HPRT-DNA: canine Hypoxanthin-Guanin-Phosphoribosyltransferase

*In collaboration with H. Murua Escobar, J. Soller

Tierärztliche Hochschule Hannover, unpublished

100bp

Neg

ativ

e co

ntro

lH

PR

T-D

NA

(12

9bp)

AuN

P-H

PR

T c

onju

gate

s*

SH


In-situ Coating with Polymers and Silica

10 nm Silica

FeOx

PVP

8 mM TEOS, NH3

(5 times)

60

65

70

75

80

85

0 20 40 60 80Dauer / [h]

D(n

50)

/ [n

m]

TEOS 8 mMTEOS 4 mM 100 nm

SilicaPVPFeOx

S. Barcikowski, S. Petersen, A. Schnoor, unpublished


In-situ Bioconjugation during laser ablation...

...enables the generation of pure bioconjugates

...is a rapid (single step) preparation method

...allows a size-tuning of bioconjugate

...offers the possibility of screening various nanoparticle-bioconjugates

...Multifunctionality ???


Thank you [email protected]

Contributions from Group 'Nanomaterials' of LZHStephan BarcikowskiNiko BärschAnnette BarchanskiAnne HahnJurij Jakobi Ana MenendezChristin MennekingLaszlo SajtiSvea PetersenRamin SattariAndreas SchwenkeArne SchnoorPhilipp WagenerJürgen WalterJohanna Walter

Financial support• DFG-Excellence Cluster REBIRTH

Junior Research Group 'Nanoparticles'

Contributions from TIHO (I. Nolte)J. Soller H. Murua Escobar


In organic Solvents, long pulses may cause pyrolysis

ControlGC/MS Control

Nd:YAG Laser, 800 nst=30min, r=5kHz, f=100mm, V=3mL, E=1.5mJ

GC/MS Control

fs-Laser, 100 fst=30min, r=5kHz, f=100mm, V= 3mL, E=300µJ

fs pulses

sub-µs/ns pulses


Ultrashort laser pulses give pure structures

Equilibrum temperature,heat conduction

Ablation with nanosecond pulses

0 s 10-15 s 10-12 s 10-9 s 10-6 s 10-3 s

10 ns Laser Pulse Duration

Photons-Electrons

Electrons-Atoms

Heat Conduction

25 Nanosecondpulse laser ablated hole in silicon

No thermal load,multiphoton effects

Ablation with femtosecond pulses

0 s 10-15 s 10-12 s 10-9 s 10-6 s 10-3 s

120 fs Laser Pulse Duration

Electrons-Atoms (Lattice)

Photons-Electrons

150 Femtosecondpulse laser ablated hole in silicon


Selecting Appropriate Laser Ablation Methods for Nanoparticle Production

Picosecond Laser Ablation• Ablation of Bulk Material in Liquids and Gases,

but: stability issues

Femtosecond Laser Ablation• (Stoichiometric) Ablation of Bulk Material

(Metals, Metal Oxides, Alloys) in Liquids and gases

IR-Laser Ablation (Continuous Wave)• Generation of 50 – 500 nm Aerosols (e.g. Metals, Polymers)

Qu

alit

y a

nd

Vers

ati

lity

Pro

du

ctivity

IR-Laser Ablation (ns pulsed)• Generation of nanoarticles,

but: thermal load of liquid and target


Bioconjugation

0 100 200 300 400 5000

2

4

6

8

10

12

14

16

18

20

22

24

26

Au-NP

Laser and Process ParametersAverage Laser Power: 0.5 W Repitition Rate: 5 kHzFocal Distance: 40 mmConcentration: 32 g/ml

Particle Diameterd90 = 64 nm

d50 = 45 nm

Re

lativ

e P

art

icle

Nu

mb

er

Fre

qu

en

cy

PN

(%

)

Hydrodynamic Diameter dh (nm)

0 100 200 300 400 5000

2

4

6

8

10

12

14

16

18

20

22

24

26

28

Au-NP in 13 µg/mL HS-DNA

Laser and Process ParametersAverage Laser Power: 0.5 W Repitition Rate: 5 kHzFocal Distance: 40 mmConcentration: 58 g/ml

Particle Diameterd90 = 52 nm

d50 = 35 nm

Re

lativ

e P

art

icle

Nu

mb

er

Fre

qu

en

cy

PN

(%

)

Hydrodynamic Diameter dh (nm)

AuSH

SH

In situ functionalization of gold nanoparticles with thiol terminated ssoligonucleotides 300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ab

sorb

an

ce [A

U]

Wavelength [nm]

Au HS-DNA HS-DNA-Au

NanoMed 2009, Berlin Nanoparticle-bioconjugates by laser ablation- a novel method aiming at pure...

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