nanomed 2009, berlin nanoparticle-bioconjugates by laser ablation- a novel method aiming at pure...
TRANSCRIPT
NanoMed 2009, Berlin
NanoMed 2009, Berlin
Nanoparticle-bioconjugates by laser ablation- a novel method aiming at
pure drug and gene deliveryS. Petersen, S. Barcikowski
JRG Nanoparticles
NanoMed 2009, Berlin
Nano Medicine
Imaging
Drug DeliveryTherapy
Targeting
Gao et al. 2004
© MagForce Nanotechnolgies
Nanobiomaterials
Science Photogallery
NanoMed 2009, Berlin
Imaging
Drug DeliveryTherapy
Targeting
Gao et al. 2004
© MagForce Nanotechnolgies
Nanobiomaterials
Science Photogallery
Core/Shell
Nanoparticle
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
Femtosecond laser ablation in liquids
• 100% pure• Material variety
• Fully dispersed and stable• Safe
fs-laser
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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)
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
Degrades the biomolecule during laser irradiation? SH
Petersen et al. Adv. Funct. Mater. 2008
NanoMed 2009, Berlin
Full integrity of biomolecules can be guaranteed
Degrades the biomolecule during laser irradiation? SH
Petersen et al. Adv. Funct. Mater. 2008
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
Petersen et al. APSUSC 2008
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
Petersen et al. Adv. Funct. Mater. 2008
Size quenching leading to monodisperse bioconjugates SH
NanoMed 2009, Berlin
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
Petersen et al. Adv. Funct. Mater. 2008
NanoMed 2009, Berlin
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
Petersen et al. Adv. Funct. Mater. 2008
NanoMed 2009, Berlin
Petersen et al. APSUSC 2008
In-situ bioconjugation with unmodified oligonucleotides
100 nm
Monodisperse Bioconjugates
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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 ???
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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
NanoMed 2009, Berlin
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