Sep 12, 2007 - Matt Hansen. Yan Zhao. Dan Zweifel. Philip Low ... Sau, T. K.; Murphy, C. J. Langmuir 2004, 20, 6414. Zweifel, D. A.; Wei, A. Chem. Mater. 2005 ...
Ligand-functionalized gold nanorods as theragnostic agents
Chemistry: Alexander Wei Matt Hansen Yan Zhao Dan Zweifel Philip Low Wei He KIST/Purdue Symposium Sep 12 2007
BME/Chem: Ji-Xin Cheng Terry B. Huff Ling Tong Haifeng Wang
Plasmon-resonant nanoparticles Surface plasmon (SP): collective excitation of conduction electrons, using light at a resonant (visible to NIR) frequency Scattering from single Au nanospheres
λinc
λem (λsca. = λinc.)
Lycurgus Cup, 4th century A.D. British Museum, London (Ag-Au NP’s embedded in glass) Absorption: red
Scattering: green
www.gold.org
Au nanoprisms: highly scattering
Au nanorods: NIR-absorbing
Gold nanorods as optical contrast agents “Biological window” in tissue at NIR wavelengths:
Au nanorods: Tunable resonances in the NIR
Attenuation is minimized between 750 nm and 1.3 μm
Transverse SP
Transmission
Absorption
(λmax) Au
Absorption
LSP
102
Absorption Coefficient
Longitudinal SP
H O 2 10
TSP 1
1 0-1 - 1
Hb O
2
1 0 -2
.2
.4
.6
.8 1 .0
2.
Wavelength (µm)
4.
6.
8.
SP modes as a function of aspect ratio: Link and El-Sayed, J. Phys. Chem. B 1999, 103, 3073
Synthesis of NIR-resonant Au nanorods Seeded growth using micellar surfactants (CTAB):
AgNO3, CTAB
Au
avg. width: 15-20 nm
aspect ratio: 3 to 4
avg. length: 40-60 nm
AuCl4, ascorbic acid 15-60 min 3-nm Au particle seeds
Sau, T. K.; Murphy, C. J. Langmuir 2004, 20, 6414. Zweifel, D. A.; Wei, A. Chem. Mater. 2005, 17, 4256-61.
Nanorod instability over time results in “optical drift”
Normalized O.D.
1x wash, 0 d 1x wash, 1 d 2x wash, 0 d 2x wash, 1 d
400
500
600
700
800
Wavelength (nm)
900
1000
Day 0: dumbbell-shaped (stabilized after Na2S)
Day 1: cylindrical
Two-photon luminescence (TPL) of Au nanorods Au nanorods
sp-hole relaxation after 2-photon abs.
TPL excitation of single nanorods
6sp fs-pulsed laser excitation
1 5d
2ν
ν’ 2
TPL excitation spectrum vs. LSP band 1.2
0.6
0.8
0.4 0.4 0.2 0.0 400 500 600 700 800 900
IPL (a.u.)
Absorbance (a.u.)
0.8
Initial polarization: α = 90° Au
0.0
Wavelength (nm)
Wang, H. et al. PNAS 2005, 102, 15752-56.
Au
Two-photon luminescence (TPL) of Au nanorods Au nanorods
sp-hole relaxation after 2-photon abs.
TPL excitation of single nanorods
6sp fs-pulsed laser excitation
1 5d
2ν
ν’ 2
TPL excitation spectrum vs. LSP band 1.2
0.6
0.8
0.4 0.4 0.2 0.0 400 500 600 700 800 900
IPL (a.u.)
Absorbance (a.u.)
0.8
Initial polarization: α = 90° Au
0.0
Wavelength (nm)
Wang, H. et al. PNAS 2005, 102, 15752-56.
Au
Single-nanorod tracking by TPL microscopy a
(c) Nanorod velocity through cell. (+) values are in the direction of the cell nucleus.
b
24.0 Y Position (μm)
(a,b) Position and trajectory of CTAB-coated nanorod through KB cell.
23.5
23.0 26.0
c
1.5
2
MSD = 4 D t + (v t) 2 D=0.00042 μm /s V = 0.023 μm/s
1.0
d
2
MSD (cm )
Speed (μm/s)
0.1
(d) Mean-square displacement (MSD) of gold nanorod, in accord with an active transport mechanism.
26.5 27.0 X Position (μm)
0.0
0.5
-0.1 0 10 20 30 40 50 60 time (s)
0.0 0
10
20 30 time (s)
40
50
Huff, T. B.; Hansen, M. N.; Zhao, Y.; Cheng, J.-X.; Wei, A. Langmuir 2007, 23, 1596-99.
in vivo TPL imaging using mouse model inject nanorods in tail
TPL imaging of blood vessel in mouse ear
see nanorods through ear Signal collected 10 min after injection at a rate of 1.12 seconds per frame. Conc. of nanorod injection: 1.7 pM TPL excitation conditions: λex = 830 nm, 200-fs pulses, 77 MHz 18 mW @ sample
hair root
175 × 175 μm
Wang, Huff, Zweifel, He, Low, Wei, and Cheng, PNAS 2005, 102, 15752-56.
in vivo TPL imaging using mouse model inject nanorods in tail
TPL imaging of blood vessel in mouse ear
see nanorods through ear Signal collected 10 min after injection at a rate of 1.12 seconds per frame. Conc. of nanorod injection: 1.7 pM TPL excitation conditions: λex = 830 nm, 200-fs pulses, 77 MHz 18 mW @ sample
hair root
175 × 175 μm
Wang, Huff, Zweifel, He, Low, Wei, and Cheng, PNAS 2005, 102, 15752-56.
in vivo TPL imaging using mouse model inject nanorods in tail
TPL imaging of blood vessel in mouse ear
see nanorods through ear Signal collected 10 min after injection at a rate of 1.12 seconds per frame. Conc. of nanorod injection: 1.7 pM TPL excitation conditions: λex = 830 nm, 200-fs pulses, 77 MHz 18 mW @ sample
Wang, Huff, Zweifel, He, Low, Wei, and Cheng, PNAS 2005, 102, 15752-56.
Targeting tumor cells with nanorods Issue #1: Nonspecific uptake of CTAB-coated Au nanorods into cells Au NR Me3N
Br
5 hours
5 days
Confocal TPL images of nanorod internalization by KB cells (no visible toxic effect after 5 days)
NMe3
Bar = 10 μm.
Nanorod-mediated photothermolysis Nanorods in cells, before cw-NIR
Br
NMe3
EtBr stained cells, after cw-NIR
Severe membrane blebbing after 30-s NIR irradiation (80 mW @ sample) Bar = 5 μm.
Huff, T. B.; Tong, L.; Zhao, Y.; Hansen, M. N.; Cheng, J.-X.; Wei, A. Nanomedicine 2007, 2, 105-12.
Targeting tumor cells with nanorods Issue #2: Control of nanorod surface chemistry Surface fouling and nonspecific protein adsorption: a serious problem Chemisorptive ligands are desorbed or displaced from Au surfaces by biogenic thiols, such as free cysteine (blood) or glutathione (intracellular). X
X
X X
X
X
+
cysteine, glutathione S
S
S
S S
Au nanoprobe surface
X+ H3N S
X H3N
O
O S S
deterioration of surface
Alternative surface functionalization: in situ Dithiocarbamate (DTC) assembly on gold Alkyl amine
DTC ligands R1
R1
N H
R2
Au surface + CS2
S
N C
R1 R2
S
N C
R2 S
S
water, DMSO, or alcohol
HO O N
N
O CH3 N
N S
S
HN
S
CH3 S
OCH3
N S
HN
NH
O S
S
S
HO NH
OH O
OH O OH
OH HO
S
OH
N S
S
Zhao, Y.; Pérez-Segarra, W.; Shi, Q.; Wei, A. J. Am. Chem. Soc. 2005, 127, 7328-29.
DTCs are resistant to surface desorption
Contact angle meas. of dibutyl-DTC monolayers on Au, before and after 24-hr exposure to ME:
OH
N S
S
diC4-DTC
S ME
XPS spectra of dibutyl-DTC 3 on Au, with and without 24-hr exposure to ME: S/N ratio constant at 2:1
Zhao, Y.; Pérez-Segarra, W.; Shi, Q.; Wei, A. J. Am. Chem. Soc. 2005, 127, 7328-29.
Controlling the cellular uptake of nanorods BSP =
Br
H3C(H2C)15 NMe3
Me3N (CH2)15CH3 Br
H2N
OCH3
O
n
CS2, pH 9.5
Au NR
S S
H N
O
OCH3 n
Preparation of mPEG-coated nanorods (n~75) by in situ DTC assembly
TPL intensity per cell (a.u.)
Au NR
SO3-
200 P
100% 150
SO3-
100
33% 50
6% 0
CTAB
BSP mPEG-DTC
Relative levels of uptake by KB cells exposed to nanorods with different coatings, after 24-h incubation.
Huff, T. B.; Hansen, M. N.; Zhao, Y.; Cheng, J.-X.; Wei, A. Langmuir 2007, 23, 1596-99.
Targeting folate receptors on KB cells Au NR
S
6 hrs
H N
S HO2C
O Folate =
O
O
N Folate H
18
O N H
O N H
N N
NH N
NH2
17 hrs Folate–oligoethyleneglycol ligands conjugated onto nanorod surfaces by in situ DTC assembly
Slower rates observed for receptormediated nanorod uptake
Huff, T. B.; Tong, L.; Zhao, Y.; Hansen, M. N.; Cheng, J.-X.; Wei, A. Nanomedicine 2007, 2, 105-12.
Site-dependent hyperthermia mediated by folate-conjugated nanorods Membrane-bound F-NRs (after 6 h) before
after, w/ EB stain
Internalized F-NRs (after 17 h) after, w/ EB stain
before
81 s scan, cw mode: fluence = 24 J/cm2
10 µm
66mw mw, CW cw before
81 s scan, fs-pulsed mode: fluence = 3 J/cm2
after, w/ EB stain
60 mw, cw before
after, w/ EB stain
0.75 mw, fs before
after
control cells (no NRs)
60 mw, cw
4.5 mw, fs
L
Threshold fluence for hyperthermic damage is 10X lower or more when nanorods are on cell membranes
Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X. Adv. Mater. 2007 19, 3136-41.
Real-time imaging of nanorod-mediated membrane blebbing in KB cells
TPL excitation conditions: λex = 765 nm, 200-fs pulses, 77 MHz, 0.75 mW @ sample Blebs are proximal and distal to membrane-bound nanorods
Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X. Adv. Mater. 2007 19, 3136-41.
Nanorod-mediated membrane blebbing due to disruption of actin filaments KB cells expressing actin-GFP
before cw irrad.
after cw irrad. (81 s, 90 mW)
Redistribution of actin-GFP; disappearance of nanorod TPL signal (melted)
internalized Au nanorods
GFP intensity after irradiation (%)
10 μm
Cyto D
100
KB cells treated with cytochalasin D (inhibitor of actin polymerization)
80 60 40 20 0
With F-NR Without F-NR
Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X. Adv. Mater. 2007 19, 3136-41.
Nanorod-mediated membrane blebbing is due to extracellular Ca2+ influx A
before B
after C
(A,B) KB cells with membrane-bound F-NRs (red) in PBS containing 0.9 mM Ca2+ (100 mg/L CaCl2) exhibited blebbing after exposure to fspulsed laser irradiation at 3 mW for 61.5 s. (C) Incubation with 2.5 µM EB (red) and 2 µM Oregon Green 488 for 20 min indicated a compromise in membrane integrity and an elevation in intracellular Ca2+, respectively. Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X. Adv. Mater. 2007 19, 3136-41.
Gold nanorods compromise membrane integrity (but cell death is chemically induced) A
before irrad. B Ca2+ free
after irrad. C Ca2+ free
after adding 1 mM Ca2+
Membrane blebbing is the direct result of actin depolymerization, induced by a change in intracellular Ca2+. (A,B) KB cells with membrane-bound F-NRs (red) in Ca2+-free PBS, before and after exposure to fs-pulsed laser irradiation at 3 mW for 61.5 s. No visible signs of damage after NIR exposure! (C) Membrane blebs appeared immediately after adding 1 mM Ca2+. Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X. Adv. Mater. 2007 19, 3136-41.
The next level 1. in vivo targeting of folate-DTC conjugated nanorods, using nude mouse tumor model 2. Application of nanorods to deep imaging modalities (photoacoustic tomography, photothermal MRI) 3. Preclinical evaluation (ADME/T) of folate-DTC conj. nanorods Microdosing for clinical evaluations, under new FDA guidelines for exploratory investigational new drugs (e-IND):
http://www.fda.gov/cder/guidance/7086fnl.htm
Conclusions - Gold nanorods are useful contrast agents for NIR imaging modalities -Nanorods can be imaged in vivo by TPL microscopy with single-particle sensitivity (also useful for optical coherence tomopgraphy (OCT)- Steve Boppart, UIUC) - Nanorods are highly efficient transducers of photothermal effects -Hyperthermic effects are most intense when nanorods are adsorbed on the cell membrane surface - Nanorod-induced cavitation causes membrane perforation, which produces a sudden influx of Ca2+ leading to membrane blebbing -in situ dithiocarbamate (DTC) formation is a simple and robust method of surface functionalization, compatible with nanomedicine applications
Sponsors NIH / NIBIB NSF / CHE Oncological Sciences Center, Purdue University
