Engineering, 2012, 5, 68-71
doi:10.4236/eng.2012.410B017 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
Ultrasound Mediated Delivery of Liposomal Doxorubicin in
Mice with Glioma
Feng-Yi Yang1, Shih-C heng H or ng2
1Department of Biomedical, Imaging and Radi o lo g ical Sciences, S chool of Biomed i cal Science and Engi neering,
Nati onal Yang-Ming University, Taipei, Taiwan
2Department of Compu ter Science & In f o r mation , Engineering, Chaoyang University of Tech nology, Taichung, Taiw an
Received 2012
Malignant brain tumors remain difficult to treat with chemotherapy because the bloodbrain barrier (BBB) limits the amounts of
pot ent agents th at can reach the tumor , such that the d rugs are un able to reach ther apeut ic dosage. Alth ough variou s targeted carriers
that encapsulate chemotherapeutic agents have been shown to improve drug delivery to brain tumors, the BBB is still a major ob-
stacle in the use of chemotherapy for the treatment of these tumors. Human glioblastoma-bearing mice were injected intravenously
with doxorubicin (Dox) encapsulated in atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes or unconjugated li-
posome. These treatments took place with or without BBB disruption induced by transcranial pulsed high-intensity focused ultra-
sound (pulsed HIFU). This study showed that the treatment with Dox encapsulated in AP-1-conjugated liposomes followed by pulsed
HIFU enhanced the accumulation of the cytotoxic drug in cells and inhibited the growth of brain tumors in vivo. Combining pulsed
HIFU with cytotoxic agents might improve their efficacy in patients with brain tumors while simultaneously reducing the drug side
effects. Further investigation is required to provide a comprehensive physical characterization of the sonication process and to deter-
mine its bioeffects.
Keywords: Focused Ult ra s ound; IL-4; Blood-b rain Barrier; Brain Tumor; Drug Delivery
1. Introduction
The limited amount of chemotherapeutic agent present in the
circulation is able to be transported into brain tumors without
the assistance of a blood-tumor barrier (BTB) delivery system
due to the presen ce of the BTB. So me works have repo rted that
using infusion of hyperosmotic solution of mannitol, which
disrupts the BTB, the drug uptake in brain tumor could be
higher than in tumor without BTB disruption [1-3]. Lipo-
some-based drug delivery systems have been designed to in-
crease tumor drug levels while limiting systemic drug dosage
[4] . It is thought that the employment of liposomes conjugated
to an tibodies o r targeti ng ligand s can enh ance the accu mulatio n
and retention of drugs at the tumor region compared with the
free drug [5] .
The glioblastoma multiforme (GBM) is one of the most
common forms of glioma. It is hard to treat gliomas completely
by surgical res ection and therefore rad iotherapy and ch emothe-
rapy are used to remove residual microscopic tumor material
[6] . Previous reports have demonstrated that high-dose chemo-
therapy may have a potential survival benefit compared to his-
torical controls treated with standard-dose therapy [7-8]. It has
been shown that human brain tumor cell lines express high
levels of pl asma membrane i nterleukin-4 receptors (IL-4R) [9].
Thus, selective drug delivery may be achievable by binding
chemotherapeutic agents to IL-4R [10]. A ligan d from atheros-
clerotic plaque-specific peptide-1 (AP-1) was selected from
phage display libraries that can locate atherosclerotic plaque
tissue and bind to the IL-4 recept or, since i t has the s ame bin d-
ing motif to the IL-4 protein. AP-1-labeled nanoparticles have
been used for the targeted drug delivery to tumor in the pre-
vious works [11-12].
The purpose of this study was to demonstrate that the tech-
nology combines pulsed HIFU and liposomal nanoparticles as a
synergistic delivery system for treating malignant brain tumors.
2. Methodology
2.1. Gl i oma Xenograft Model
Male 6 to 8-week-old NOD-scid mice were anesthetized by an
intraperitoneal administration of pentobarbital at a dose of 40
mg/kg of body weight. All animal experiments were performed
according to the appropriate guidelines and approved by our
Animal Care and Use Committee. The 2 × 105 Human brain
malignant glioma (GBM8401) cells in 2 μL culture medium
were injected into the brains of the mice. Magnetic Resonance
imaging (MRI) was used to determine that a tumor was estab-
2.2. Ultrasound Equipment
Pulsed HIFU exposures were generated by a 1.0-MHz, sin-
gle-element focused transducer (A392S, Panametrics, Waltham,
MA, USA) with a diameter of 38 mm and a radius of curvature
of 63.5 mm. T he fo cal zo ne o f the ther apeu ti c transdu cer was in
the shape of an elongated ellipsoid, with a radial diameter (–6
dB) of 3 mm and an axi al length (–6 dB) of 26 mm. The trans-
ducer with removab le co ne was fixed on a st ereo taxic ap par atu s
Copyright © 2012 SciRes. E NG
(Stoelting, Wood Dale, IL, USA) that permitting submilimeter
placement of the ultrasound focus at the target location in the
brain. A function generator (33220A, Agilent Inc., Palo Alto,
USA) was connected to a power amplifier (500-009, Advanced
Surgical Syste ms, Tucso n , AZ) to drive the FUS transducer and
a power meter/sensor module (Bird 4421, Ohio, USA) was used
to measure the input electrical power. The rat’s head was
mounted on the stereotaxic apparatus with the nose bar posi-
tioned 3.3 mm below the interaural line. UCA (SonoVue,
Bracco International, Amsterdam, The Netherlands) was in-
jected into the femoral vein of the rats about 15 s before each
sonication. The UCA contains phospholipid-coated microbub-
bles with a mean diameter = 2.5 μm, and at a concentrat ion o f 1
x 108 to 5 x 108 bubbles/ml. Sonication was pulsed with a burst
length of 50 ms at a 5% duty cycle and a repetition frequency
of 1 Hz. The duration of the sonication was 60 s. The ultra-
sound beam was delivered to one location in the left brain he-
misphere, centered on the tumor injection site. The following
sonication parameters were used: an acoustic power of 2.86 W
with an injection of 300 μl/kg UCA, a pulse repetition fre-
quency of 1 Hz, and a duty cycle of 5%.
2.3. Preparation of Liposomal Doxorubicin
Lip o-Dox was prepared using a solvent injection method plus
remote loading procedures. Briefly, hydrogenated soybean l-α-
phosphatidylcholine (95.8 mg, Avanti Polar Lipids), cholesterol
(31.9 mg, Sigma-Aldrich), and 1,2 -distearoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]
(DSPE-PEG2000, 31.9 mg, Avanti Polar Lipids) were dis-
solved and well mixed in 1 ml of absolute ethanol at 60ºC. The
lipid and ethanol mixture was then injected into a 9 -ml solution
of 250 mM ammonium sulfate and stirred for 1 h at 60ºC. The
mixture was then extruded five times through polycarbonate
membranes (Isopore Membrane Filter, Millipore) with pore
sizes of 0.4, 0.2, 0.1, and 0.05 µm, consecutively, at 60ºC with
high-pressure extrusion equipment (Lipex Biomembranes) to
produce small liposomes. The liposome suspension was then
dialyzed five times against large amounts of 10% sucrose con-
taining 5 mM NaCl to remove the unentrapped ammonium
sulfate and ethanol. After dialysis, the liposome suspension was
placed in a 50-ml glass bottle in a 60ºC water bath and mixed
with Dox, to a final Dox concentration of 2 mg/ml in 10% su-
crose solution. The bottle was intermittently shaken in a 60ºC
water bath for 1 h and then immediately cooled down to 4ºC,
culminating in the production of Lipo-Dox.
Due to the presence of a thiol group on each cystine of the
AP-1 peptide (CRKRLDRNC), it is possible to couple AP-1 to
liposomes via the thiol-maleimide reaction. Briefly, AP-1 pep-
tide was conjugated to
1,2-distearoyl-sn-g lycer o-3-phosphoethanolamine-N-[maleimid
e(polyethylene glycol)-2000] (DSPE-PEG2000-MAL, Avanti
Polar Lipids) by adding AP-1 to the DSPE-PEG2000-MAL
micelle solution at a 2:1 molar ratio while mixing at 4ºC over-
night. The free thiol groups were measured with
5,5’-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, Sig-
ma-Aldrich) at 420 nm to confirm that most of the AP-1 was
conjugated with DSPE-PEG2000-MAL after the reaction.
AP-1-conjugated DSPE-PEG2000 was transferred into the pre-
formed Lipo-Dox at a 1.5% molar ratio of total lipid compo-
nents and incubated at 60ºC for 1 h to obtain AP-1-labeled
Lip o-Dox (AP-1 Lipo-Dox; F igure 1).
2.4. Quantitative Evaluation of Doxorubicin
The procedures of single and repeated sonication are shown in
Figure 2. The concentration of liposomes administered to the
mice via tail-vein injection corresponded to 5 mg/kg. The brain
was perfused by transcardiac methods with normal saline 3.5 h
after the Dox administration in order to flush unabsorbed Dox
from the cerebral vessels. The site of tumor tissue was har-
vested alo ng with its contralateral count erpart as a control. The
concentration of Dox present was measured using a spectro-
photometer (PowerWave 340, BioTek, USA; excitation at 480
nm and emissions measured at 590 nm), with the value deter-
mined by ta king the average o f at least th ree fluoro metric read-
ings. The Dox present in the tissue samples was quantified
using a linear regression standard curve derived from seven
different concentrations of Dox; the amount of Dox was quanti-
fied as the absorbance per gram of tissue.
2.5. Treatment Protocol
A group of control mice was injected with GBM8401 glioma
cells, but received no treatment. Five and 9 days after tumor
cell implantation, one group of glioma-bearing mice received
one of the following: (1) Lipo-Dox, or (3) AP-1 Lipo-Dox fol-
lowed by single sonication. Another group of glioma-bearing
mice treated Lipo-Dox or AP-1 Lipo-Dox followed by repeated
sonication on day 5 after tumor implantation.
Figure 1. Schematic diagram for the AP-1 liposomal doxorubicin.
Liposomes were prepared containing maleimide-functionaled po-
lyethylene glycol chains. The maleimide was used to attach the
AP-1 peptide through the thiol group on a cystine.
Copyright © 2012 SciRes. ENG
Figure 2. Diagrams of the experimental time line for (A) single
sonication and (B) repeated sonication.
2.6. MR Imaging
Magneti c resonance i maging (MR I) was performed us ing a 3-T
MRI system (TRIO 3-T MRI, Siemens MAGNETOM, Ger-
many) after focused ultrasound sonication. The mice were
anesthetized with isoflurane mixed with oxygen during the
imaging procedure. A loop coil (Loop Flex Coil, approximately
4 cm in diameter) was used for RF reception. The imaging
plane was located across the center of the tumor injection site.
Tumor progression was monitored by means of T2-weighted
images obtained from 4 to 16 days after tumor implantation.
The parameters for T2-weighted imaging were as follows: re-
petition time/echo time = 3500/75 ms, matrix = 125×256, field
of view = 25×43 mm, and secti on thi ckness = 1.0 mm
3. Results
Figure 3 shows the mean concentration of Dox per unit mass
for the contralateral normal brain, the brain tumors and the
brain tumors with single or repeated sonication after unconju-
gated Lipo-Dox or AP-1 Lipo-Dox administration.
The concentration of Dox was not only significantly greater
in the unsonicated tumor BBB than in the contralateral normal
brain region, but it was also significantly greater at the tumor
site after single or repeated sonication than in the unsonicated
tumor for the two forms of Lipo-Dox, especially for the re-
peated sonication. In addition, the concentration of Dox was
significantly greater at the tumor site with unconjugated
Lip o-Dox followed by single or repeated sonication than in the
unsonicated tumor treated with AP-1 Lipo -Dox without sonica-
Figure 3. Measurements of Lipo-Dox and AP-1 Lipo-Dox in con-
tralateral normal brain, the brain tumor and the brai n tumor with
single or repeated sonication. Compared with the contralateral
normal brain, there was a significant differe nce for the control and
sonicated tumors between the two forms of the drug. The concen-
trations of Dox were significantly higher in sonicated brai n tumors
than in unsonicated brain tumors. */***, #/### and ††/††† denote
significant differences compared with the contralateral normal
brain, the unsonicated brain tumor and single sonicated tumor,
respectively. (*,#,P<0.05, ††P<0.01, ***,###, †††P<0.001; n=3 mice per
Compared to the control tumor, there was a significant in-
crease in the derived tumor-to-contralateral brain ratios for the
single or repeated sonicated tumor treated with either drug
(Figure 4). Interestingly, the derived tumor-to-contralateral
brain ratio was significantly greater after single or repeated
sonication for the untargeted Lipo-Dox group than for the tar-
geted Lipo-Dox group without sonication.
Figure 5 shows the effects of the various treatment protocols
on tumor growth were monitored by MR imaging on day 12
after implantation. These results suggest that AP-1 Lipo-Dox
enhan ced by pulsed HI FU at the tumor site i s more effective at
inhibiting tumor growth than AP-1 Lipo-Dox alone.
4. Discussion
Malignant glioma remains one of the most deadly types of tu-
mor in humans, and GBM is one of the most common forms of
glioma. Pulsed HIFU induced high-dose chemotherapy may
have a poten tial survi val benefit compared to his torical cont rols
treated with standard-dose therapy. This study showed that the
treatment with Dox encapsulated in AP-1-conjugated liposomes
followed by single or repeated pulsed HIFU enhanced the ac-
cumulation of the cytotoxic drug in cells and inhibited the
growth of brain tumors in rats. Combining pulsed HIFU with
c yt otoxic agents might improve their efficacy in brain tumors
with minimal systemic toxicity. This technology, employed
with recent advances in targeted microbubbles, may promote
Figure 4. The derived tumor-to-contralateral brain ratios with
unsonication, single sonication and repeated sonic ation after drug
administration. (*,#P<0.05, ##,††P<0.01, ***,†††P<0.001; n=3 mice per
Figure 5. Representative sample of T2-weighted magnetic reson-
ance imaging of a human GMB 8401 xenograft on day 12 after
i mplantati on. Compared to the control tumor, there was a clear
Copyright © 2012 SciRes. E NG
decrease in the size of the tumor in mice treated with AP-1 Lipo-
Dox fo llowed by single pulsed HIFU or repeated pulsed HIFU.
new approaches to be developed that make targeted brain tumor
therapy possible. The results of this pilot study therefore sug-
gest it would provide targeted access for chemotherapy and
allow the use of recombinant pharmaceuticals for the brain
5. Acknowledgements
This study was supported by grants from the National Science
Council of Taiwan (no. NSC 100-2321-B-010-010 and NSC
99-2321-B-010-017), Cheng Hsin General Hospital Foundation
(no. 100F117CY25), Veterans General Hospitals University
System of Taiwan Joint Research Program (#VGHUST100-G1-
3-3 and V100E6-007), Yen Tjing Ling Medical Foundation
(grant CI-100-17), Department of Health of Taiwan (DOH101-
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