We develop a novel dual-modal contrast agent—encapsulated-ink poly(lactic-co-glycolic acid) (PLGA) microbubbles and nanobubbles—for photoacoustic and ultrasound imaging. Soft gelatin phantoms with embedded tumor simulators of encapsulated-ink PLGA microbubbles and nanobubbles in various concentrations are clearly shown in both photoacoustic and ultrasound images. In addition, using photoacoustic imaging, we successfully image the samples positioned below 1.8-cm-thick chicken breast tissues. Potentially, simultaneous photoacoustic and ultrasound imaging enhanced by encapsulated-dye PLGA microbubbles or nanobubbles can be a valuable tool for intraoperative assessment of tumor boundaries and therapeutic margins

Kim, Chulhong

Qin, Ruogu

Wang, Lihong V.

Xu, Jeff S.

Xu, Ronald

Caltech Authors - Main

M
m
n
p
u
C
L
a
B
B
b
E
C
A
a

t
e
m
a
i
c
1
n
e
c
t
P
K
t
P
J
F
m
t
t
e
m
H
m
i
b
b
t
d
d
T
*
W
L
l
†
O
4
JBO Letters
J
Downloaded Froultifunctional
icrobubbles and
anobubbles for
hotoacoustic and
ltrasound imaging
hulhong Kim,a Ruogu Qin,b Jeff S. Xu,b
ihong V. Wang,a,* and Ronald Xub,†
Washington University in St. Louis, Department of
iomedical Engineering, Optical Imaging Laboratory, One
rookings Drive, St. Louis, Missouri 63130
The Ohio State University, Department of Biomedical
ngineering, 270 Bevis Hall, 1080 Carmack Road,
olumbus, Ohio 43210
bstract. We develop a novel dual-modal contrast
gent—encapsulated-ink polylactic-co-glycolic acid
PLGA microbubbles and nanobubbles—for photoacous-
ic and ultrasound imaging. Soft gelatin phantoms with
mbedded tumor simulators of encapsulated-ink PLGA
icrobubbles and nanobubbles in various concentrations
re clearly shown in both photoacoustic and ultrasound
mages. In addition, using photoacoustic imaging, we suc-
essfully image the samples positioned below
.8-cm-thick chicken breast tissues. Potentially, simulta-
eous photoacoustic and ultrasound imaging enhanced by
ncapsulated-dye PLGA microbubbles or nanobubbles
an be a valuable tool for intraoperative assessment of
umor boundaries and therapeutic margins. © 2010 Society of
hoto-Optical Instrumentation Engineers. DOI: 10.1117/1.3302808
eywords: microbubbles; nanobubbles; photoacoustic imaging; ul-
rasound imaging; tumor margin intraoperative assessment.
aper 09505LR received Nov. 13, 2009; revised manuscript received
an. 1, 2010; accepted for publication Jan. 6, 2010; published online
eb. 18, 2010.
Surgical resection followed by radiation therapy and che-
otherapy is the most effective treatment for malignant in-
racranial tumors.1,2 Yet accurate localization of tumors and
heir boundaries is a critical issue with this method. Intraop-
rative magnetic resonance imaging MRI is currently the
ost reliable tool to identify the structural features of tumors.
owever, MRI alone is not sufficient to delineate diffuse tu-
or margins, because it cannot identify molecular character-
stics at the cellular level.3,4 Ultrasound US imaging has
een applied as an alternative for tumor resection control,
ecause this imaging modality is relatively inexpensive, por-
able, and fast enough to provide real-time images.5 However,
ue to its low contrast, speckle artifacts, and high operator
ependence, the reliability of US imaging is not fully proven.6
aking advantage of strong endogenous and exogenous opti-
Address all correspondence on photoacoustic imaging to: Lihong V. Wang,
ashington University in St. Louis, Department of Biomedical Engineering, St.
ouis, MO 63130. Tel: 314-935-6152; Fax: 314-935-7448. E-mail:
hwang@biomed.wustl.edu
Address all correspondence on micro- and nanobubbles to: Ronald Xu, The
hio State University, Department of Biomedical Engineering, Columbus, OH
3210. Tel: 614-688-3635; Fax: 614-292-7301. E-mail: xu.202@osu.eduournal of Biomedical Optics 010510-
m: http://biomedicaloptics.spiedigitallibrary.org/ on 07/01/2016 Terms of Usecal contrasts, pure optical imaging has been intraoperatively
applied to map tumor margins.7 However, due to strong light
scattering, pure optical imaging suffers from either shallow
penetration depth optical microscopy8 or poor spatial reso-
lution diffuse optical tomography9. Along with the applica-
tion of new imaging techniques, contrast agents have been
intensively investigated to improve the degree of resection
and preserve normal tissues.10–12 Therefore, there is a pressing
need to develop a highly sensitive and high resolution imag-
ing tool with proper contrast agents for monitoring surgical
resection of intracranial tumors.
Thanks to its strong optical absorption contrast and high
ultrasonic resolution, photoacoustic PA imaging has become
a popular technique in tumor imaging in vivo.13 The principle
of this technique is that acoustic waves are generated through
thermoelastic expansion when targets absorb a short-pulsed
light.14,15 The acoustic waves propagate in biological tissues
and are detected by ultrasound transducers. The initial pres-
sure rise is proportional to the local optical absorption the
product of the local optical absorption coefficient and the lo-
cal optical fluence. The locations of optically absorptive tar-
gets can be measured using the arrival times of ultrasound.
Since the spatial resolution at depths beyond one optical
transport mean free path 1 mm is determined by acoustic
parameters, the resolution and imaging depth can be scalable,
while pixel numbers, defined as the ratio of the imaging depth
to the resolution, are maintained at greater than 100. In addi-
tion, a conventional ultrasound system can be easily com-
bined with PA imaging to provide both mechanical contrast
morphological information and optical contrast morpho-
logical and functional information.
In this work, for the first time to our knowledge, we have
demonstrated a novel multifunctional contrast agent for PA
and US imaging—encapsulated-ink polylactic-coglycolic
acid PLGA microbubbles MBs and nanobubbles NBs.
We successfully imaged gelatin-based encapsulated-ink
PLGA MBs and NBs at various concentrations using both PA
and US imaging systems. Moreover, deeply positioned
1.8 cm below biological tissues encapsulated-ink PLGA
MBs and NBs were clearly imaged using the PA imaging
system. These results show that encapsulated-dye PLGA MBs
and NBs can potentially be employed to assess intracranial
tumor boundaries and therapeutic margins intraoperatively us-
ing simultaneous PA and US imaging.
Encapsulated-ink PLGA 50:50, RG 502H 12000 Da MW,
Boehringer Ingelheim, Germany MBs and NBs were fabri-
cated using a modified double-emulsion process.16 First, four
solutions were prepared: 1. 5 mL of 2.5% w/v PLGA solution
of methylene chloride CH2Cl2, Fisher Scientific, Worcester,
Massachusetts, 2. 50 mL of 4% w/v polyvinyl alcohol PVA,
Fisher Scientific aqueous solution; 3. 0.5 mL of 50% v/v ink
Higgins Fountain Pen India nonwaterproof black ink, San-
ford, Chicago, Illinois and 4% w/v PVA aqueous solution;
and 4. 100 mL of 5% v/v isopropanol aqueous solution. After
that, the 0.5-mL aqueous solution of ink and PVA was added
to the 5-mL PLGA solution of CH2Cl2, and emulsified in an
ice bath. Then, the emulsion was added drop-wise using a
1000-L pipette to the 50-mL PVA solution in an ice bath
and emulsified again. The double emulsion was then added to
1083-3668/2010/151/010510/3/$25.00 © 2010 SPIEJanuary/February 2010  Vol. 1511
: http://spiedigitallibrary.org/ss/TermsOfUse.aspx
t
r
w
d
w
f
C
w
u
c

5
g
H
t
O
9
c
s
9
d
t
5
0
g
p
N
l
a
t
d
w
M
2
e
f
d
c
c
p
d
t

fi
a
t
a
t
a
i
a
s
F
t
o
1
1
2
JBO Letters
J
Downloaded Frohe isopropanol solution, stirred for 1.5 h by a magnetic stir-
er, and then centrifuged. After centrifugation, the supernatant
as discarded, and the bubble precipitate was washed by
eionized water. The process of centrifugation and washing
as repeated three times. The washed spheres were then
reeze-dried by a Lyph-lock 4.5 freeze-dry system Labcono
orporation, Kansas City, Missouri for 36 h. Dried bubbles
ere harvested and stored in a glass bottle at 0 °C for further
se. For the MBs, the first and second emulsion steps were
onducted with a model 17105 Omni Mixer homogenizer
Omni International, Kennesaw, Georgia at 20,000 rpm for
min and 10,000 rpm for 3 min, respectively. The centrifu-
ation was carried out with a Centrifuge 5810R Eppendorf,
amburg, Germany at 1500 rpm for 10 min. For the NBs,
he first and second emulsion steps were conducted with an
mni-Ruptor 250 ultrasound probe Omini International at
0 W for 2 min and at 30 W for 1 min, respectively. The
entrifugation was carried out with a Sorvall RC-5B high-
peed centrifuge Sorvall, Waltham, Massachusetts at
000 rpm for 10 min.
Eight cylindrical MB and NB tumor simulators, 5 mm in
iameter, were fabricated by dispersing MBs and NBs respec-
ively in gelatin solution at four different concentrations 2.5,
, 10, and 15 mg /mL. The gelatin solution concentration:
.15 g /mL was preheated by a microwave oven and de-
assed by a vacuum pump in advance. A tissue simulating
hantom was fabricated by casting the cylindrical MB and
B tumor simulators in a gelatin pad of 340.5 cm.
The tissue simulating phantom was imaged by a 5-MHz
inear array US probe Terason, Burlington, Massachusetts
nd a PA macroscope. The schematic of PA macroscope and
he mechanisms of the ring-shaped light illumination are fully
escribed in Ref. 17. To irradiate the phantoms, we used the
avelength-tunable Ti-sapphire laser LT-2211A, Lotis TII,
insk, Belarus, pumped by a Q-switched Nd:YAG laser LS-
137, Lotis with a pulse duration of 6.5 ns and a pulse rep-
tition rate of 10 Hz. The light fluence on the phantom sur-
ace was 3.4 mJ /cm2, within the current ANSI limit.18 A
oughnut-shaped light =767 nm, formed by concave and
onical lenses, traveled through an optical condenser, and was
oaxially focused with the ultrasound focal zone in water. The
hantoms were positioned under a water tank that had a win-
ow with a transparent thin membrane. The PA waves, de-
ected by a single-element 5-MHz ultrasound transducer
V308, Panametrics-NDT, Waltham, Massachusetts, were
rst amplified and then transferred to an oscilloscope. The
xial and lateral resolutions were 138 and 490 m, respec-
ively. One-dimensional depth-sensitive images along the z
xis referred to as A-lines were obtained by measuring the
imes of arrival of the PA signals. By scanning the samples
long the x and y directions, 3-D PA images were formed. 3-D
mages were projected into 2-D images through maximum
mplitude projection MAP, which projected the strongest
ignal along each A-line onto the corresponding x-y plane.
A schematic of encapsulated-ink MBs and NBs is shown in
ig. 1a. The volume-weighted size distributions, defined as
he multiplication of the number distribution and the volume
f particles, of MBs and NBs are plotted in Figs. 1b and
c, respectively. The average size of MBs is
.010.73 m, and that of NBs is 0.290.09 m. Figures
a–2c are photographic, PA, and US images acquired fromournal of Biomedical Optics 010510-
m: http://biomedicaloptics.spiedigitallibrary.org/ on 07/01/2016 Terms of Usethe same gelatin phantom. Targets 1 through 4 are tumor
simulators made of encapsulated-ink MBs at concentrations
of 2.5, 5.0, 10, and 15 mg /mL, respectively. Targets 5
through 8 are tumor simulators composed of encapsulated-ink
NBs at concentrations of 2.5, 5.0, 10, and 15 mg /mL, respec-
tively. All eight targets are clearly shown in the PA images
Fig. 2b and matched well with the photograph. In the cor-
responding US image Fig. 2c, although targets 1 and 2 are
not clearly shown, possibly owing to multiple factors includ-
ing US depth of focus and/or low concentration of contrast
agent, the other six targets are clearly visible. The PA signals
are quantified as a function of concentration in Fig. 2d. As
the concentration mounts, the PA signals increase linearly.
Since the average size of MBs was bigger than that of NBs,
the MBs encapsulated more ink, and consequently PA signals
Fig. 1 a Schematic of MBs and NBs encapsulating black India ink.
The size distributions of encapsulated-ink b MBs and c NBs. The x
axis is a log scale. VWD: Volume weighted distribution.
Fig. 2 a Photograph of a phantom containing tumor simulators
made of encapsulated-ink MBs and NBs with various concentrations.
b The corresponding PA image. c The corresponding US image. d
The quantification of the PA signals at various concentrations of MBs
and NBs. e The quantification of the US signals at various concen-
trations of MBs and NBs. 1 through 4: MBs at concentrations of 2.5,
5.0, 10, and 15 mg/mL, respectively. 5 through 8: NBs at concentra-
tions of 2.5, 5.0, 10, and 15 mg/mL, respectively.January/February 2010  Vol. 1512
: http://spiedigitallibrary.org/ss/TermsOfUse.aspx
f
s
c
t
h
g
p
f
i
i
a
m
l
s
g
c
i
e
e
c
e
P

c
b
c
b
m
s
A
T
I
C
t
D

p
w
F
m

8
p
t
r
1
JBO Letters
J
Downloaded Frorom MBs were stronger than those from NBs. Figure 2e
hows a linear increase of the US signals as a function of
oncentration. Note that the US signals in NBs are stronger
han those in MBs. The enhanced acoustic contrast by NBs
as not been well studied yet, possibly due to particle aggre-
ation and the layering effect.19,20 These results strongly sup-
ort the dual functionality of encapsulated-ink MBs and NBs
or PA and US imaging, where the optical contrast stems from
nk and the mechanical contrast from bubbles.
To take advantage of the deeper penetration depth of PA
maging with high resolution, we added chicken breast tissues
top the phantom. The phantom was prepared in the same
ethod as mentioned before Fig. 3a. With an 8-mm-thick
ayer of chicken breast tissue, all eight targets are clearly
hown in Fig. 3b. With an 18-mm-thick layer, although tar-
ets 1, 2, 5, and 6 are relatively dim, targets 3, 4, 7, and 8 are
learly visible in Fig. 3c. This result implies that PA imaging
s potentially able to delineate deep tumor margins using the
ncapsulated-ink MBs or NBs.
In summary, we successfully developed and tested
ncapsulated-ink PLGA MBs and NBs as a dual-modality
ontrast agent for PA and US imaging. Deeply positioned
ncapsulated-ink MBs and NBs were clearly imaged by the
A imaging. In the future, clinically practical organic dyes
such as indocyanine green encapsulated in MBs and NBs
an be tested in PA imaging.16 By treating the surface of the
ubbles to target specific molecules, the applications of these
ontrast agents using simultaneous PA and US imaging can be
roadened to include not only intracranial tumor boundary
apping but also molecular imaging of primary and meta-
tatic tumor.21
cknowledgments
his work was supported in part by grants from the National
nstitutes of Health R01 EB000712, R01 EB008085, R01
A113453901, U54 CA136398—the Network for Transla-
ional Research, and 5P60 DK02057933 to Wang, and the
epartment of Defense Breast Cancer Research Program
W81XWH-07 to Xu. Wang has a financial interest in Micro-
hotoacoustics, Incorporated, and in Endra, Incorporated,
hich, however, did not support this work.
ig. 3 a Photograph of a phantom containing tumor simulators
ade of encapsulated-ink MBs and NBs with various concentrations.
b The corresponding PA image of the phantom positioned below
mm of chicken breast tissues. c The corresponding PA image of the
hantom positioned below 18 mm of chicken breast tissues. 1
hrough 4: MBs at concentrations of 2.5, 5.0, 10, and 15 mg/mL,
espectively. 5 through 8: NBs at concentrations of 2.5, 5.0, 10, and
5 mg/mL, respectively.ournal of Biomedical Optics 010510-
m: http://biomedicaloptics.spiedigitallibrary.org/ on 07/01/2016 Terms of UseReferences
1. M. Ammirati, N. Vick, Y. L. Liao, I. Ciric, and M. Mikhael, “Effect
of the extent of surgical resection on survival and quality of life in
patients with supratentorial glioblastomas and anaplastic astrocyto-
mas,” Neurosurgery 212, 201–206 1987.
2. M. A. Hammoud, B. L. Ligon, R. elSouki, W. M. Shi, D. F. Schomer,
and R. Sawaya, “Use of intraoperative ultrasound for localizing tu-
mors and determining the extent of resection: a comparative study
with magnetic resonance imaging,” J. Neurosurg. 845, 737–741
1996.
3. P. C. Johnson, S. J. Hunt, and B. P. Drayer, “Human cerebral gliomas:
correlation of postmortem MR imaging and neuropathologic find-
ings,” Radiology 1701, 211–217 1989.
4. C. Nimsky, O. Gansland, M. Buchfelder, and R. Fahlbusch, “Intra-
operative visualization for resection of gliomas: the role of functional
neuronavigation and intraoperative 1.5 T MRI,” Neurol. Res. 285,
482–487 2006.
5. G. Unsgaard, O. M. Rygh, T. Selbekk, T. B. Muller, F. Kolstad, F.
Lindseth, and T. A. Hernes, “Intra-operative 3D ultrasound in neuro-
surgery,” Acta Neurochir. 1483, 235–253 2006.
6. M. Gerganov, A. Samii, A. Akbarian, L. Stueglitz, M. Samii, and R.
Fahlbusch, “Reliability of intraoperative high-resolution 2D ultra-
sound as an alternative to high-field strength MR imaging for tumor
resection control: a prospective comparative study,” J. Neurosurg.
1113, 512–519 2009.
7. R. Trehin, J. L. Figueiredo, M. J. Pittet, R. Weissleder, L. Josephson,
and U. Mahmood, “Fluorescent nanoparticle uptake for brain tumor
visualization,” Neoplasia 84, 302–311 2006.
8. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson,
W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. P. Puliafito, and J. G.
Fujimoto, “Optical coherence tomography,” Science 2545035,
1178–1181 1991.
9. B. W. Zeff, B. R. White, H. Dehghani, B. L. Schlaggar, and J. P.
Culver, “Retinotopic mapping of adult human visual cortex with
high-density diffuse optical tomography,” Proc. Natl. Acad. Sci.
U.S.A. 10429, 12169–12174 2007.
10. M. M. Haglund, M. S. Berger, and D. W. Hochman, “Enhanced op-
tical imaging of human gliomas and tumor margins,” Neurosurgery
382, 308–317 1996.
11. M. M. Haglund, D. W. Hochman, A. M. Spence, and M. S. Berger,
“Enhanced optical imaging of rat gliomas and tumor margins,” Neu-
rosurgery 355, 930–940 1994.
12. M. F. Kircher, U. Mahmood, R. S. King, R. Weissleder, and L. Jo-
sephson, “A multimodal nanoparticle for preoperative magnetic reso-
nance imaging and intraoperative optical brain tumor delineation,”
Cancer Res. 6323, 8122–8125 2003.
13. L. V. Wang, “Multiscale photoacoustic microscopy and computed
tomography,” Nature Photon. 39, 503–509 2009.
14. A. Rosencwaig and A. Gersho, “Theory of the photoacoustic effect
with solids,” J. Appl. Phys. 471, 64–69 1976.
15. M. Xu and L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev.
Sci. Instrum. 774, 041101 1–22 2006.
16. R. Xu, J. Huang, J. Xu, D. Sun, G. Hinkle, J. Edward, W. Martin, and
S. Povoski, “Fabrication of indocyanine green encapsulated biode-
gradable microbubbles for structural and functional imaging of can-
cer,” J. Biomed. Opt. 143, 034020 2009.
17. K. H. Song and L. V. Wang, “Deep reflection-mode photoacoustic
imaging of biological tissue,” J. Biomed. Opt. 26, 060503 2007.
18. American National Standard for Safe Use of Lasers ANSI Z136.1–
2000, Laser Institute of America, Orlando, FL 2000.
19. J. Liu, J. Li, T. J. Rosol, X. Pan, and J. L. Voorhees, “Biodegradable
nanoparticles for targeted ultrasound imaging of breast cancer cells in
vitro,” Phys. Med. Biol. 5216, 4739–4747 2007.
20. J. N. Marsh, K. C. Partlow, D. R. Abendschein, M. J. Scott, G. M.
Lanza, and S. A. Wickline, “Molecular imaging with targeted per-
fluorocarbon nanoparticles: quantification of the concentration depen-
dence of contrast enhancement for binding to sparse cellular
epitopes,” Ultrasound Med. Biol. 336, 950–958 2007.
21. J. S. Xu, J. Huang, R. Qin, G. Hinkle, S. Povoski, E. Martin, and R.
X. Xu, “Synthesizing and binding dual-mode poly lactic-co-glycolic
acid PLGA nanobubbles for cancer targeting and imaging,” Bio-
materials 317, 1716–1722 2010.January/February 2010  Vol. 1513
: http://spiedigitallibrary.org/ss/TermsOfUse.aspx


Multifunctional microbubbles and nanobubbles for photoacoustic and ultrasound imaging

https://authors.library.caltech.edu/70200/1/010510_1.pdf

Multifunctional microbubbles and nanobubbles for photoacoustic and ultrasound imaging

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main