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Carbon nanoparticles as a
multimodal thermoacoustic and
photoacoustic contrast agent
Xin Cai, Lina Wu, Wenxin Xing, Jun Xia, Liming Nie, et
al.
Xin Cai, Lina Wu, Wenxin Xing, Jun Xia, Liming Nie, Ruiying Zhang,
Gregory M. Lanza, Baozhong Shen, Dipanjan Pan, Lihong V. Wang, "Carbon
nanoparticles as a multimodal thermoacoustic and photoacoustic contrast
agent," Proc. SPIE 8581, Photons Plus Ultrasound: Imaging and Sensing
2013, 858140 (4 March 2013); doi: 10.1117/12.2005064
Event: SPIE BiOS, 2013, San Francisco, California, United States
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Carbon Nanoparticles as a Multimodal Thermoacoustic and
Photoacoustic Contrast Agent
Xin Cai,
1
Lina Wu,
2,3
Wenxin Xing,
1
Jun Xia,
1
Liming Nie,
1
Ruiying Zhang,
1
Gregory M. Lanza,
2
Baozhong Shen,
3
Dipanjan Pan,
2
and Lihong V. Wang
1,
∗
1
Optical Imaging Laboratory, Depart
ment of Biomedical Engineeri
ng, Washington University in St.
Louis, St. Louis, Missouri 63130
2
Division of Cardiology, Washington University
School of Medicine, St. Louis, MO 63108
3
Molecular Imaging Center
and Radiology Department, 4
th
Affiliated Hospital of Harbin Medical
University, Harbin, China
ABSTRACT
We demonstrated the potential of carbon nanoparticles (CNP
s) as exogenous contrast ag
ents for both thermoacoustic
(TA) tomography (TAT) and photoacoustic (PA) tomography
(PAT). In comparison to deionized water, the CNPs
provided a four times stronger signal in TAT at 3 GHz.
In comparison to blood, The CNPs provided a much stronger
signal in PAT over a broad wavelength range of 450–850 nm. Specifically, the maximum signal enhancement in PAT
was 9.4 times stronger in the near-infrared window of 635–670 nm.
In vivo
blood-vessel PA imaging was performed
non-invasively on a mouse femoral area. The images, captur
ed after the tail vein injection of CNPs, show a gradual
enhancement of the optical abso
rption in the vessels by up to 230%. The results indicate that CNPs can be potentially
used as contrast agents for TAT and PAT
to monitor the intravascular or extravascu
lar pathways in clinical applications.
Keywords:
Contrast agents; carbon; photoacoustic tomogra
phy; thermoacoutic tomography; nanoparticle.
1. INTRODUCTION
Thermoacoustic (TA) tomography (TAT) and photoacoustic (PA) tomography (PAT) are non-invasive techniques that
uniquely synergize pure ultrasound and pure radio frequency (rf) and optical imaging, allowing both satisfactory spatial
resolution and high soft-tissue contrast.
1-2
The technique is based on the detection of acoustic waves from an object that
absorbs pulsed or intensity-modulated el
ectromagnetic energy (rf band in TAT and laser in PAT). The absorption can be
associated with endogenous molecules, such as water/ion, hemoglobin, and melanin. For instance, due to the high
concentration of hemoglobin (12 to 15 g/dl), blood inhe
rently has a strong optical absorption which allows the
visualization of blood vessels. However, the absorption of nonvascular tissues (
e.g.
, lymph nodes) or intravascular
biosignatures (
e.g.
, integrins) is insufficient. Thus, exogenous contrast agents such as optical dyes, gold nanoparticles,
copper nanoparticles, and carbon nanotubes are needed for TAT/PAT in these cases.
3-6
TAT and PAT have been
developed for different applications in rodent models, such as breast cancer imaging, brain structural and functional
imaging, blood oxygenation and flow velocity monitoring, and tumor angiogenesis.
1,7-8
∗
Corresponding author:
lhwang@biomed.wustl.edu
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 858140 · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2005064
Proc. of SPIE Vol. 8581 858140-1
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Although a lot of contrast agents are available for TAT and
PAT, they have their disadvantages. For example, optical
dyes easily cause skin staining; gold nanoparticles are high in
cost and have complicated chemistries; and the toxicity of
copper nanoparticles and carbon nanotubes is arguable. Ther
efore, new contrast agents still deserve exploration. We
reveal a simple and commercially amen
able synthetic methodology for creating optically active carbon nanoparticles
(CNP). CNPs are derived from commercial food-grade honey. Compare to the previously explored particles (gold,
copper, carbon nanotube
etc.
), CNPs are significantly smaller (~8 nm in
diameter), enabling rapid clearance properties.
In this study, we explore the rf and optical absorbtion properties of carbon nanoparticles (CNPs) as multimodal contrast
agents for both TAT and PAT. Then, we further monitor the pharmacokinetics of CNPs in blood vessels in mice
in vivo
using PAT. The results could be greatly beneficial for m
onitor the intravascular or ex
travascular pathways using
TAT/PAT together with other structural imaging modalities.
2. MATERIALS AND METHODS
2.1 Synthesis of optically ac
tive carbon nanoparticles
Commercial grade honey (Great Value
™
Clover Honey 1 wt%; batch composition- fructose: 38%, glucose: 31%,
maltose: 7.1%, sucrose: 1.3%, higher sugars: 1.5%, water: 17.2%) is suspended with an organic macromolecular
passivating agent (8wt%; PEG400), purged with argon, and heated in a domestic microwave oven for 30 min.
Microwave power was set at 1200 W with an output power
of 50%. The product was th
en purified by repeated
centrifugation in water.
2.2 Thermoacoustic system
For thermoacoustic excitation, a 3.0-GHz microwave generator with pulse width of 0.6
μ
s and repetition rate of 10 Hz
was employed. The pulses were guided toward the target thr
ough a horn antenna. The size of the antenna opening was 11
cm
×
7 cm. The fluence is 0.45 mJ/cm
2
at the opening of the antenna, which is within the safety standard. The test
samples were placed in a plastic tank filled with mineral
oil for ultrasonic coupling.
A 1-MHz spherically focused
transducer with a bandwidth of 70% (V314, Panametr
ics, Olympus) was used to receive TA signals.
2.3 Photoacoustic system
The schematic of the system has been reported before.
9
For photoacoustic excitation, three different light sources were
employed for different spectra range or imaging speed: 1) a tunable OPO laser (450–685 nm, Vibrant (HE) 355 I,
OPOTEK), pulse width 5 ns, pulse repetition rate 10 Hz; 2)
a tunable Ti:sapphire laser
(730–850 nm, LT-2211A, LOTIS
TII) pumped by a Q-switched Nd:YAG (L
S-2137/2, LOTIS TII), pulse width <15 ns, pulse repetition rate 10 Hz; and 3)
a dye laser (CBR-D, Sirah) pumped by
a Nd:YLF laser (INNOSLAB, Edgewave),
pulse width <7 ns, pulse repetition
rate up to 5 kHz. The first two sources were used for the
measurement of the PA spectr
um of CNPs. The third source
was used for fast PA imaging
in vivo
. The fluence is < 1 mJ/cm
2
on the sample surface. For photoacoustic detection, a
focused ultrasonic transducer with 50 MHz central fre
quency (V214-BB-RM, Olympus
NDT) was employed. The
transducer surface was immersed in wate
r for ultrasonic c
oupling. The optical
and ultrasonic foci were configured
coaxially and confocally. This
system could achieve 45
μ
m lateral resolution, 15
μ
m axial resolution, and more than 3
mm penetration depth.
2.4 Animals
All animal experiments were performed in accordance with protocols approved by the Washington University
Department of Comparative Medicine and the Animal Studies Committee. Athymic nude mice were obtained from
Harlan and housed in the animal facility at Washington University. During the experiments, the animals were
anesthetized by administration of gaseous isoflurane
(2%, Butler Inc., Dublin)
and aseptically prepared.
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a
bA
.7 -2
Q
1-
20n
)
s
:NPs
Vater
18
Time (p
n
20
.$)
2
Fig. 1. (a) A t
y
dispersed stat
e
As-synthesi
z
From the sc
a
(number-ave
r
Water and i
o
Therefore, t
o
of water. Fi
g
volume) fill
e
signal.
10
At
(Fig. 2).
Fig. 2. TA sig
n
y
pical photogra
p
e
.
z
ed CNPs are
s
a
nning electro
n
r
aged) of 8 ±
2
o
ns are two w
o
show that C
N
g
. 2a shows th
e
e
d with CNPs
3 GHz, CNP
s
n
als at 3 GHz fr
o
p
h of solution o
s
oluble in wat
e
n
microscopy (
S
2
n
m
.
ell-known so
u
N
Ps can functi
o
e
TA signals
o
(33 vol%) or
s
(33 vol%) e
x
o
m a LDPE vial
f CNPs in wate
r
3.
R
e
r. Fig. 1a sho
w
S
EM) image (
F
u
rces of micro
w
o
n as a contras
t
o
btained from
a
deionized (D
I
x
hibited more
(I.D. = 6 mm a
n
r
(33 vol%). (b)
R
ESULTS
w
s a typical p
h
F
ig. 1b), it is
c
w
ave absorbe
r
t
agent for TA,
a
low-density
p
I
) water. The
than fou
r
-fol
d
n
d 1.5 cc volum
e
A scanning ele
c
h
otograph of s
c
lear that CNP
s
r
s in human,
a
we first comp
p
olyethylene (
LDPE vial d
o
d
TA signal e
n
e) filled with C
N
ctron microsco
p
s
olution of CN
s
are spherical
a
nd they gene
r
p
ared the TA s
i
(
LDPE) vial (I
.
o
es not genera
t
n
hancement co
N
Ps (33 vol %)
a
p
y image of CN
P
Ps in water (3
and exhibit a
r
ate strong T
A
i
gnal of CNPs
w
.D. = 6 mm a
n
te any measu
r
o
mpared with
D
a
nd DI wate
r
P
s in dry-
3 vol%).
diameter
A
signals.
w
ith that
n
d 1.5 cc
r
able TA
D
I water
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+r
"I
CO u
Ce
...moo
...moo
z
R
4
)
450
55
Wi
n
-/
0
650
welengtN
o
PA ampli
750
1
(nm)
L
LL
tude (a. u
_ _
I
à
850
IM
1
0
Di
4
ar
6
3
b
o
-3
6l
16
fferential
nplitude (
I
17
Time
Bic
f\
CN
18
(μs)
lod
Ps
"
19
Similarly, h
e
efficacy of
C
CNPs (33 v
o
signal. Fig.
3
excitation w
a
The PA sign
which cover
e
exhibited 9.
4
Fig. 3. (a) Ra
t
signals gener
a
Preliminary
wavelength
w
4a). After th
e
taken at the
s
clarity than
t
differential i
m
differential
o
Fig. 4. Nonin
v
before the inj
e
Differential i
m
e
moglobin is a
C
NPs with blo
o
o
l %) and rat
w
3
a shows the
a
velength ran
g
a
l obtained fr
o
e
d both visibl
e
4
times PA sig
n
t
io of the pea
k
-
t
t
ed from CNPs
a
in vivo
blood
-
w
as tuned to 6
e
CNPs (25 v
o
s
ame position
w
t
hat in Fig. 4
a
m
age in Fig. 4
o
ptical absorpt
i
v
asive PA ima
g
e
ction of CNPs.
m
age that was o
b
dominant opt
i
o
d at various
e
w
hole blood.
T
ratio of the
p
g
e was 450–8
5
o
m CNPs was
e
and near inf
r
n
al enhanceme
n
o-peak PA sign
a
a
nd rat bloo
d
at
vessel PA im
a
50 nm. Befor
e
o
l %, 100 μL)
w
ith an interv
a
a
d
id, because
c is a result o
f
i
on in the vasc
u
g
ing the femora
l
(b) PA image
a
b
tained by subtr
a
i
cal absorber i
n
e
xcitation wa
v
T
he tube is tr
a
p
ea
k
-to-
p
eak P
5
0 nm, excludi
n
much stronge
r
r
ared (NIR) li
g
n
t compared t
o
a
ls generated fr
o
the wavelength
a
ging was per
f
e
CNP injectio
n
were intrave
n
a
l of 1–2 min.
the exogenou
s
f
the subtractio
n
u
la
r
caused
b
y
l
area of a mo
u
a
cqui
r
ed at 6 m
i
a
cting the pre-in
j
n
humans and
v
elengths. PA
s
a
nsparen
t
, and
A signal amp
l
n
g the range
o
r
than or comp
g
h
t
. For exam
p
o
blood (Fig. 3
b
o
m CNPs (33 v
o
of 670 nm.
f
ormed non-i
n
n
, a PA imag
e
n
ously injecte
d
At 6 min post
-
s
contrast age
n
n
of Fig. 4a fr
o
the exogenou
s
u
se
in vivo
emp
l
i
n post-injectio
n
j
ection image (a
)
generates stro
n
s
ignals were
o
therefore, it
d
litude of CN
P
o
f 685–730 n
m
p
arable to
b
loo
d
p
le, at the exc
b).
o
l %) to those
o
n
vasively on a
e
of the femor
a
d
through the t
a
-injection (Fi
g
n
t, CNPs, ind
u
o
m Fig. 4b. T
h
s
contras
t
age
n
l
oying CNPs as
n
of CNPs (25
v
) from the post-
i
n
g PA signals
.
o
btained from
t
d
oes not produ
P
s (33 vol %)
m
due to the g
a
d
over such b
r
itation
λ
=670
o
f bloo
d
at diffe
mouse femor
a
a
l was acquire
d
a
il vein,
a ser
i
g
. 4b), the vasc
u
ced optical a
b
h
is image indi
c
n
t.
contrast agent
s
v
ol %, 100 μL)
i
njection image
.
We compare
d
t
ygon tubes fi
l
ce any measu
r
to that of bl
o
a
p between t
w
r
oad waveleng
t
nm, CNPs (3
3
e
rent wavelengt
h
a
l area. The e
x
d
as the base-
l
i
es of PA ima
g
c
ulature showe
d
b
sorption incr
e
c
ates the distri
b
s
. (a) PA imag
e
through the tai
l
(b).
d
the PA
l
led with
r
able PA
o
od. The
w
o lasers.
t
h range,
3
vol %)
h
s. (b) PA
xcitation
l
ine (Fig.
g
es were
d
greater
e
ase The
b
ution of
e
acquired
l
vein. (c)
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4. CONCLUSION
In summary, we have successfully demonstrated the potential of carbon nanoparticles (CNPs) as exogenous contrast
agents for both thermoacoustic tomography (TAT) and photoacoustic tomography (PAT). The CNPs provided more than
4 times signal enhancement in TAT at 3 GHz and more than 9.4 times signal enhancement in PAT in the NIR window of
635–670 nm. The results indicate that CNPs can be potentiall
y used as contras agents for TAT and PAT together with
other structural imaging modalities to monitor the intravascular or extravascular pathways in clinical applications.
5. ACKNOWLEDGMENTS
Our work was sponsored by NIH grants R01 EB000712, R01 EB008085, R01 CA140220, R01 CA157277, R01
CA159959, U54 CA136398, and DP1 EB016986—NIH Director’s Pioneer Award. L.V.W. has a financial interest in
Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work. Others claim no competing
financial interests.
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