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Nuclear Medicine and Biology 36 (2009) 515 – 524

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[186 Re]Liposomal doxorubicin (Doxil): in vitro stability,

pharmacokinetics, imaging and biodistribution in a head and neck
squamous cell carcinoma xenograft model
Anuradha Soundararajana , Ande Baoa,b , William T. Phillipsa ,
Ricardo Perez III a , Beth A. Goinsa,⁎
a
Department of Radiology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
b
Department of Otolaryngology—Head and Neck Surgery, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
Received 19 December 2008; received in revised form 14 February 2009; accepted 18 February 2009

Abstract

The purpose of this study was to determine the feasibility of radiolabeling liposomal doxorubicin (Doxil) for cancer chemoradionuclide
therapy by directly loading the therapeutic radionuclide rhenium-186 (186Re) into the liposome interior. The pharmacokinetics, imaging and
biodistribution of [186Re]Doxil (555 MBq/kg) and control [186Re]polyethylene glycol (PEG) liposomes (555 MBq/kg) were determined after
intravenous administration in a head and neck cancer xenograft model in nude rats. [186Re]Doxil and [186Re]PEG liposomes were
radiolabeled using [186Re]N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine. 186Re labeling efficiency was 76.1±8.3% with Doxil. The
in vitro serum stability of [186Re]Doxil at 37°C was 38.06±12.13% at 24 h. Pharmacokinetic studies revealed that [186Re]Doxil had a two-
phase blood clearance with half clearance times of 0.8 and 28.2 h. Images acquired over 120 h showed that [186Re]Doxil had slow blood
clearance, low liver accumulation and increasing spleen accumulation. The biodistribution study at 120 h indicated that the percentage of
injected dose (%ID) in the blood and tumor for [186Re]Doxil was 20-fold higher than that of [186Re]PEG liposomes. The %ID values in the
kidney and liver were not significantly different between [186Re]Doxil and [186Re]PEG liposomes. These results suggest that the long
circulation and prolonged bioavailability of [186Re]Doxil could potentially deliver high concentrations of both doxorubicin and 186Re to
tumor when encapsulated in the same liposome vehicle.
© 2009 Elsevier Inc. All rights reserved.

Keywords: Rhenium-186; Doxil; Biodistribution; Pharmacokinetics; SPECT/CT; Liposome; Nanoparticle

1. Introduction preferentially accumulated in tumor xenografts and human

tumors. Doxil had a 20- to 30-h blood half clearance time in
Liposomal doxorubicin (Doxil) is a liposome formulation tumor-xenograft-bearing mice and rats and a 50- to 60-h half
that encapsulates the chemotherapeutic drug doxorubicin, clearance time in humans [1–3]. Doxil has been approved for
thereby decreasing its toxicity [1]. An ammonium sulfate the treatment of AIDS-related Kaposi's sarcoma, recurrent
(pH) gradient is used to load and stably retain doxorubicin in ovarian cancer and metastatic breast cancer [2,4].
the liposome interior. Doxil also contains polyethylene Head and neck squamous cell carcinoma (HNSCC)
glycol (PEG) lipid, resulting in pegylation of the liposome accounts for nearly 5% of cancer cases in the United States
surface, which enables it to have prolonged circulation and [5]. Most HNSCC patients present with advanced-stage
reduced volume of distribution, thereby improving tumor disease (Stage III/IV) [6]. Surgery and radiation therapy are
uptake through enhanced permeability and retention (EPR) the primary treatment modalities. Although good prognosis
effect and by extending effective tumor therapy [1,2]. has been achieved for early-stage disease (Stage I/II), the 5-
Animal and human studies have shown that Doxil year survival rates are only 30–40% for late-stage disease
due to local recurrence and distant metastases [7]. Combina-
tion therapy with various treatment modalities has been
⁎ Corresponding author. Tel.: +1 210 567 5575; fax: +1 210 567 5549. developed to improve survival and to maintain critical
E-mail address: goins@uthscsa.edu (B.A. Goins). functions [8]. Concomitant administration of chemotherapy
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.02.004
516 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524

and radiation therapy may enhance tumor cytotoxicity from Committee. All experimental procedures were conducted
radiation, resulting in improved locoregional tumor therapy, while the animals were anesthetized with 1–3% isoflurane
but at the cost of increased normal tissue toxicity [7,9]. High (Vedco, St. Joseph, MO) in 100% oxygen using an
local drug concentration is required to prevent local anesthesia inhalation unit (Bickford, Wales Center, NY).
recurrence; this can be achieved by using liposomes as A previously characterized human head and neck cancer
drug delivery systems, as they have improved pharmacoki- xenograft model in nude rats was used [24]. SCC-4 cell line
netic profile in comparison to free drugs. Preclinical and (ATCC, Manassas, VA) was cultured and maintained at
clinical studies with Doxil have shown improved efficacy in 37°C in an incubator with 5% CO2. When the cells were
head and neck tumors [10,11] and enhancement of radio- 80–90% confluent, they were collected and made into a
therapy effect [12]. single cell suspension in saline. Male rnu/rnu athymic nude
Liposomes have been labeled with diagnostic and rats (Harlan, Indianapolis, IN) age 4–5 weeks (75–100 g)
therapeutic radionuclides [13]. Preclinical studies of lipo- were inoculated subcutaneously with 5×106 of SCC-4
somes labeled with 186 Re [14–16] and 188Re [17–19] have tumor cells in 0.20 ml of saline on the dorsum at the level
been reported. Theoretical dosimetry studies have suggested of the scapulae. Tumor dimensions were determined by
that liposomes with therapeutic radionuclides, when admi- measuring the length (l), width (w) and depth (d) of each
nistered intravenously, would deliver a high radiation tumor using digital calipers. Tumor volume was calculated
absorbed dose to the tumor while sparing the bone marrow using the ellipsoid volume formula: V=(π/6)lwd [25].
and controlling liver and spleen doses to acceptable levels Animals were used for the study when tumor volume was
[20,21]. 186Re is an attractive radionuclide for imaging and ∼1.5 cm3, which typically occurred between 15 and 16 days
therapy because of its 3.78-day half-life, with a 137-keV γ after tumor cell inoculation.
emission for scintigraphic imaging and a β emission of a
maximum of 1.07 MeV energy with a tissue penetration 2.2. Preparation of liposomes
depth range of 2–4 mm for tumor therapy [22].
Doxil uses an ammonium sulfate gradient to load and Doxil, a commercially available liposomal doxorubicin
retain doxorubicin in liposomes, and the same gradient can formulation manufactured by Johnson&Johnson (New
be used to load and trap diagnostic radionuclide, technetium- Brunswick, NJ), was purchased from Oak Hills Pharmacy
99m (99mTc) and the therapeutic radionuclides 186Re and (San Antonio, TX). Doxil contains 2 mg/ml doxorubicin,
188
Re with a complex of N,N-bis(2-mercaptoethyl)-N′,N′- 3.19 mg/ml N-(carbonyl-methoxypolyethylene glycol
diethylethylenediamine (BMEDA) [19,23]. By trapping 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine
186
Re in Doxil, both doxorubicin and 186 Re will be carried sodium salt (DSPE-PEG 2000), 9.58 mg/ml fully hydro-
in the same liposome for combination chemoradionuclide genated soy phosphatidylcholine and 3.19 mg/ml choles-
therapy. Although biodistribution of intravenously adminis- terol. Control PEG liposomes containing ammonium sulfate
tered [99mTc]Doxil has been studied in normal rats [23], pH gradient were manufactured in-house with lipid compo-
therapeutic applications of [186Re]Doxil in head and neck sition and particle diameter in reference to Doxil's lipid
cancer xenografts have not been reported yet. PEG composition and particle size (Table 1). PEG liposomes
liposomes with similar lipid composition and particle size containing 1,2-distearoyl-sn-glycero-phosphatidylcholine
and with ammonium sulfate gradient were prepared as a (DSPC; Avanti Polar Lipids, Pelham, AL), DSPE-PEG
control. Prior to initiation of chemoradionuclide therapy 2000 (Avanti Polar Lipids) and cholesterol (Calbiochem, San
studies, the in vitro stability, pharmacokinetics and biodis- Diego, CA) (weight ratio 3:1:1) were manufactured follow-
tribution of intravenously administered [186Re]Doxil and ing a previously reported method with minor modifications
[186Re]PEG liposomes were investigated. The distribution of [14,26]. Lipid mixture dissolved in a chloroform/methanol
both formulations in nude rats with head and neck xenografts mixture (2:1 vol/vol) [27] was dried to form a lipid thin film
was followed for 5 days postadministration using micro by rotary evaporation and desiccated overnight. The lipid
single-photon emission tomography (SPECT)/computed film was rehydrated with 300 mM sucrose (Ferro Pfanstiehl
tomography (CT). In this study, the feasibility of preparing Laboratories, Cleveland, OH) in sterile water for injection,
[186Re]Doxil with high efficiency and its in vitro serum warmed to 60°C for complete suspension of lipids and
stability, pharmacokinetics, imaging and biodistribution in
an HNSCC model are demonstrated. Table 1
Characteristics of Doxil and control PEG liposomes
Doxil PEG liposomes
2. Materials and methods
Diameter (nm) 87.3±8.5 91.3±11.8
2.1. Animal model Phospholipid (mg/ml) 9.58 17.47
DSPE-PEG 2000 (mg/ml) 3.19 4.71
All animal experiments were conducted in accordance Cholesterol (mg/ml) 3.19 4.71
with the National Institutes of Health Animal Use Guidelines Ammonium sulfate gradient (mM) 250 240
Total lipid (mg/ml) 15.96 26.89
and with prior approval of our Institutional Animal Care
 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524 517

lyophilized overnight. The dried lipid–sucrose mixture was agarose gel (2.0 ml) was packed in a microcolumn (Bio-Rad,
rehydrated with 240 mM ammonium sulfate (Sigma, St. Hercules, CA) by centrifugation at 1500 rpm for 2 min.
Louis, MO) in sterile water then subjected to five freeze– Then, 10 column volumes of PBS (pH 7.4) were used to
thaw cycles at 60°C, followed by extrusion through a series equilibrate the column. At the desired time points (1, 4, 24,
of polycarbonate filters (2 μm, 400 nm, 200 nm, 2 passes 48, 72 and 96 h), 100 μl of [186 Re]Doxil/[186Re]PEG
each; 100 nm, 5 passes; 50 nm, 10 passes) (Lipex Extruder liposome serum solution was added to the equilibrated spin
and Whatman Nucleopore filters; Northern Lipids, Vancou- column and centrifuged at 1500 rpm for 1 min, and the first
ver, Canada). The extruded liposome solution was stored at fraction was collected in a tube. Then, 100 μl of PBS (pH
4°C until needed. 7.4) was added to the column and centrifuged at 1500 rpm
The diameters of Doxil and control PEG liposomes were for 1 min, and the second fraction was collected in a new
measured with a 488-nm laser light scattering instrument tube. This elution process was repeated 19 times, and each
(Brookhaven Instruments, Holtsville, NY). Phospholipid fraction was collected in a new tube after centrifugation. The
content was measured for control PEG liposomes using 186
Re activity in each fraction was counted using a Minaxi γ
Stewart assay [28]. Control PEG liposomes were checked for A5550 γ counter (Perkin-Elmer Life and Analytical
bacterial growth and pyrogenicity (University Hospital Sciences, Boston, MA). The percentage of 186Re activity
Pathology Laboratory, San Antonio, TX). No bacterial associated with Doxil/PEG liposomes was calculated by
growth was detected within a 14-day culture, and endotoxin summing the activity in the first eight fractions divided by
level was b5 EU/ml. the total activity in all 20 fractions. The above procedure was
repeated with [186Re]Doxil and [186 Re]PEG liposomes
2.3. Preparation of [186Re]Doxil/[186Re]PEG liposomes stored in PBS (pH 7.4) at 25°C at the same time points (1,
4, 24, 48, 72 and 96 h).
To a vial containing 50 mg of glucoheptonate (GH;
Sigma-Aldrich, St. Louis, MO) and 3.0 μl of BMEDA
(ABX, Radeberg, Germany), 2.0 ml of nitrogen-degassed 2.5. Biodistribution and pharmacokinetic studies
saline was added. The mixture was mixed by magnetic
Fourteen male nude rats with HNSCC xenografts were
stirring for 20 min, followed by addition of 240 μl of freshly
used for this study. [186Re]Doxil (n=7) or [186Re]PEG
prepared stannous chloride solution (15 mg/ml). An aliquot
liposomes (n=7) were intravenously injected through the
of 1.0 ml of the GH–BMEDA–stannous chloride mixture
tail vein at a dose of 555 MBq/kg (15 mCi/kg) under
was placed in a new vial after adjusting the pH of the mixture
anesthesia. The total lipid dose for both groups was
to 5.0. The vial was flushed with nitrogen and sealed. [186 Re]
maintained at 52 mg/kg. The doxorubicin dose was
Perrhenate solution [∼2.96 GBq (80 mCi); University of
maintained at 6.5 mg/kg for the [186 Re]Doxil group. Rats
Missouri Research Reactor, Columbia, MO] was added to
were sacrificed at 120 h postinjection by cervical dislocation.
the vial and incubated at 80°C for 1 h. After incubation, the
All major organs and tissues were collected in 10% buffered
[186 Re]BMEDA solution was cooled to 25°C before
formalin (Fisher Scientific, Pittsburgh, PA). The organs were
adjusting the pH of the solution to 7.0. Immediately before
weighed and counted for 186Re activity using a Wallac
being used for radiolabeling, Doxil or PEG liposomes were
Wizard automatic γ counter (Perkin-Elmer Life and Analy-
eluted with phosphate-buffered saline (PBS; pH 7.4) through
tical Sciences). A 50-μl standard of [186Re]Doxil or [186Re]
a PD-10 column (GE Healthcare, Piscataway, NJ) to create
PEG liposomes was also counted and used for decay
an ammonium sulfate pH gradient by removing free
correction. Data are expressed as percentage of injected
ammonium sulfate from the liposome exterior. Eluted
dose per gram (%ID/g) and percentage of injected dose per
Doxil or PEG liposomes were added to [186Re]BMEDA
organ (%ID/organ).
solution and incubated at 37°C for 1 h. Finally, labeled
The pharmacokinetics of [186 Re]Doxil and [186Re]PEG
[186 Re]Doxil or [186 Re]PEG liposomes were separated from
liposomes was determined by collecting blood samples in a
free [186 Re]BMEDA by eluting through PD-10 columns
microcentrifuge tube through the tail vein at 0.08 (5 min),
with PBS (pH 7.4). Labeling efficiency was calculated by
0.5, 1, 2, 4, 8.5, 24, 48, 72, 96 and 120 h after intravenous
dividing 186 Re activity in Doxil/PEG liposomes after
injection. The weight of each blood sample was determined
separation by the total 186Re activity before separation.
by weighing the microcentrifuge tube before and after blood
2.4. In vitro labeling stability studies collection. The concentrations of radioactivity in the blood
were calculated as %ID/g, and %ID/g at 5 min was
An aliquot of the final [186 Re]Doxil/[186 Re]PEG lipo- normalized to 100%. The blood clearance patterns of
some sample was added to an aliquot of fetal bovine serum [186Re]Doxil and [186 Re]PEG liposomes were simulated
(FBS; Invitrogen, Carlsbad, CA) in a 1:1 volume ratio, and using Origin software, version 7.5 (Origin Lab, North-
the solution was incubated at 37°C. The amount of 186 Re ampton, MA). A dual-exponential equation, Y=b1e−c1t +b2-
activity associated with the liposomes at different time points e−c2t, was used. Here Y is %ID in blood; t is the time after
was measured using an ACL 4% cross-linked agarose gel injection; and b1, b2, c1 and c2 are constants. The two-phase
(Sooner Scientific, Garvin, OK) spin column [23,29]. The blood clearance half-times [(t1/2)1 and (t1/2)2] were calculated
518 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524

from the simulated dual-exponential curves as follows: (t1/ micrometer-thick sections of each tumor specimen were
2)1=0.693/c1 and (t1/2)2=0.693/c2 [23]. prepared and stained with hematoxylin and eosin (H&E) for
histopathological examination and comparison with the
2.6. Micro-SPECT/CT imaging and image analysis autoradiography images.
Re has a penetrative 137-keV γ emission that allows
186
2.8. Statistical analysis
for the monitoring of the biodistribution of labeled liposomes
using γ scintigraphy. High-resolution parallel hole collima- The data are presented as mean±standard deviation (S.D.).
tor (HRES) planar γ camera images and SPECT images were Group comparisons were performed with analysis of
acquired in a 137(±10%)-keV energy window using a micro- variance, using Origin software (Origin Lab). Pb.05 was
SPECT scanner equipped with dual cadmium zinc telluride considered significant.
detectors (FLEX SPECT/CT/PET; Gamma Medica, North-
ridge, CA). Static planar images were acquired in two views
3. Results
(anterior–posterior and lateral) at baseline and 4, 20, 46, 70,
96 and 118 h after [186Re]Doxil/PEG liposome injection. An 3.1. Labeling efficiencies
∼0.26-MBq (∼70 μCi) standard source of [186Re]Doxil or
[186Re]PEG liposomes was placed in the field of view, but The labeling efficiencies of [186Re]Doxil and [186 Re]PEG
outside the position of the rat during static planar image liposomes were 76.1±8.3 % (n=4) and 77.1±8.4% (n=4),
acquisition for image quantification. Tomographic images respectively. There was no significant difference in labeling
with parallel hole collimators (32 projections; 7000 counts/ efficiencies for [186Re]Doxil and [186 Re]PEG liposomes
projection) were acquired at the same time points as the when either 4.44 or 2.22 GBq of 186Re activity was used for
planar static images and also acquired with multipinhole liposome labeling.
(MPH) collimators [32 projections; 7000 counts/projection;
3.2. In vitro stability studies
radius of rotation=5 cm; field of view=9.78 cm] at 20 h.
SPECT images reconstructed using the Lumagen processing The in vitro stabilities of [186 Re]Doxil and [186 Re]PEG
software available with the system had a matrix size of liposomes following incubation in FBS at 37°C are shown
80×80×80 and a voxel dimension of 1.6 mm. CT images in Fig. 1A. The 186Re activity associated with Doxil was
were acquired at 20 h and reconstructed at a matrix size of 80.42±4.27% at 4 h and 12.25±1.67% at 48 h. The 186 Re
512×512×512 with a 0.17-mm voxel dimension using the activity associated with PEG liposomes was 81.28±4.44%
software available with the scanner. and 6.06±1.78% at 4 and 48 h, respectively. The 186 Re
The planar images acquired at each time point were activity associated with Doxil and PEG liposomes after
analyzed to determine %ID/g values in the blood, liver, storage in PBS (pH 7.4) at 25°C is shown in Fig. 1B. After
spleen, kidneys, intestines and tumor. Region of interest the storage of [186 Re]Doxil and [186 Re]PEG liposomes at
(ROI) was drawn around the standard source to obtain 25°C in PBS (pH 7.4) for 24 h, there was 26.39±4.00% and
counts to activity (mCi) conversion factor. To determine % 40.25±5.44% of 186Re activity associated with Doxil and
ID/g in tumor at 4 h, ROI was drawn over the tumor in PEG liposomes, respectively.
lateral images, and the counts obtained were converted into
activity. Using the weight of the tumor obtained at 120 h 3.3. Pharmacokinetic studies
after biodistribution and injected activity, %ID/g in tumor at
The blood clearance curves of [186Re]Doxil and [186 Re]
4 h was determined. No blood pool correction was applied.
PEG liposomes from baseline to 120 h are shown in Fig. 2.
The %ID/g values for the listed organs at each time point for
The maximum radioactivities in the blood were determined as
both [186Re]Doxil and [186Re]PEG liposomes were deter-
2.64±0.09%ID/g and 3.22±0.39%ID/g at 0.08 h for [186 Re]
mined as above.
Doxil and [186 Re]PEG liposomes, respectively. [186 Re]Doxil
2.7. Autoradiography and histopathology showed a slow blood clearance, with 1.31±0.07%ID/g
remaining in the blood at 24 h. In contrast, [186 Re]PEG
From the tumor excised at 120 h, a thin slice (∼1 mm) liposomes showed a more rapid clearance, with 1.66±0.12%
was sectioned along the longest dimension of the tumor for ID/g and 0.11±0.01% ID/g remaining in the blood at 0.5 and
autoradiography. The thin section was placed on a reusable 24 h, respectively. Exponential curve-fitting analysis of the
phosphor imaging plate (DenOptix; Gendex Dental Sys- clearance curves showed a two-phase blood clearance for
tems, Lake Zurich, IL) at −20°C to obtain an autoradio- both [186Re]Doxil and [186 Re]PEG liposomes. For [186 Re]
graphy image. The plate was exposed to the tumor section Doxil, 17.04% of the injected activity had a half clearance
for 2 h for [186Re]Doxil and for 20 h for [186Re]PEG time of 0.8 h, and 83.2% of the injected activity had a half
liposomes. The latent image was converted into a digital clearance time of 28.2 h. For [186Re]PEG liposomes, 84.01%
image by laser photostimulation scanning (Gendex Dental of the injected activity was cleared with a half clearance time
Systems). The same tumor section was then fixed in 10% of 0.42 h, and 14.9% of the injected activity had a half
buffered formalin for 48 h and embedded in paraffin. Four- clearance time of 18.6 h. The half clearance time for [186 Re]
 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524 519

Fig. 2. Blood clearance curves of [186Re]Doxil and [186Re]PEG liposomes

after intravenous administration in HNSCC tumor-bearing rats. Data are
presented as mean %ID±S.D. (n=7 at each time point for each group).

showed slow blood clearance indicated by a high level of

radioactivity in the heart, low liver uptake and high spleen
accumulation. [186Re]Doxil also had a consistently high

Fig. 1. In vitro stability of [186Re]Doxil and [186Re]PEG liposomes at

different times after incubation in FBS at 37°C (A) or in PBS (pH 7.4) at
25°C (B) (mean±S.D.; n=3).

Doxil is similar to the reported half clearance time for Doxil

[2], suggesting that the in vivo stability of [186Re]Doxil might
be better than that of in vitro incubation. The half clearance
time of [186 Re]Doxil was significantly longer than that of
[186 Re]PEG liposomes. This long circulation time could
deliver a higher concentration of [186 Re]Doxil into the tumor,
thereby improving tumor therapy by chemotherapy from
doxorubicin and radionuclide therapy from 186Re.

3.4. Micro-SPECT/CT imaging and planar image analysis

Micro-SPECT/CT images of [186 Re]Doxil and [186 Re]

PEG liposomes acquired using a MPH collimator at 20 h
after administration are shown in Fig. 3. Prolonged blood
retention, decreased liver uptake and increased tumor
uptake were observed in images of rats receiving [186 Re] Fig. 3. Micro-SPECT/CT images acquired at 20 h postadministration of
[186Re]Doxil using a MPH collimator. A three-dimensional volume-
Doxil. Lateral planar scintigraphic images of [186 Re]Doxil rendered SPECT image of [186Re]Doxil overlaid with CT isosurface
and [186Re]PEG liposomes acquired at different time points displayed in the bone window shows accumulation in the tumor (T), liver
after intravenous injection are shown in Fig. 4. [186 Re]Doxil (L), spleen (S) and circulation through the heart (H).
520 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524

Fig. 4. Lateral planar scintigraphic images depicting the distribution of [186Re]Doxil (top) and [186Re]PEG liposomes (bottom) at various time points after
injection. The slow clearance of [186Re]Doxil, low accumulation in liver and high accumulation in tumor are seen. (H, heart; L, liver; S, spleen; K, kidney; T,
tumor; STD, standard).

level of accumulation in the abdominal region. In

comparison, [186Re]PEG liposomes showed a more rapid
blood clearance and high liver and spleen accumulation.
[186Re]Doxil and [186Re]PEG liposomes had a similar
excretion pattern through the kidneys. The %ID/g values in
tumor and other organs at different time points determined
from image analyses for [186 Re]Doxil and [186Re]PEG
liposomes are shown in Fig. 5. The results showed that
[186Re]Doxil had a significantly higher %ID/g value in the
blood than [186 Re]PEG liposomes at all time points (Pb.01).
The maximum radioactivity in the blood was 2.57±0.16%
ID/g and 0.52±0.05%ID/g at 4 h for [186 Re]Doxil and
[186Re]PEG liposomes, respectively. For [186Re]PEG lipo-
somes, radioactivity in the liver, spleen and tumor reached
maximum levels of 3.65±0.42%ID/g, 1.85±0.85%ID/g and
0.86±0.43%ID/g, respectively, at 4 h. For [186Re]Doxil,
radioactivity in the liver, spleen and tumor reached
maximum levels of 1.72±0.11%ID/g at 20 h, 5.24±1.86%
ID/g at 46 h and 2.06±0.52%ID/g at 4 h, respectively. The
maximum radioactivity in the kidney was 7.49±1.32%ID/g
at 46 h for [186 Re]Doxil and 8.81±2.03%ID/g for [186Re]
PEG liposomes at 20 h.

3.5. Biodistribution studies

The biodistribution values at 120 h after the administra-

tion of [186Re]Doxil and [186Re]PEG liposomes are shown in
Table 2. Comparison of the %ID/organ values for [186Re]
Doxil and [186Re]PEG liposomes showed that the radio-
activity in the blood was 1.70±1.20%ID and 0.09±0.07%ID
Fig. 5. The distribution of [186Re]Doxil (A) and [186Re]PEG liposomes (B) at
for [186Re]Doxil and [186Re]PEG liposomes, respectively. various time points, as determined from planar image analysis. The %ID/g
The %ID in the blood was 20-fold higher for [186Re]Doxil values in the blood, tumor and spleen are significantly higher for [186Re]Doxil.
than for [186Re]PEG liposomes (Pb.01). Similarly, the %ID Data are presented as mean±S.D. (n=4 at each time point and for each group).
 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524 521

Table 2 3.6. Comparison of histopathology and

Biodistribution of [186Re]PEG liposomes and [186Re]Doxil at 120 h after autoradiography images
injection (n=7 in each group)
Organ %ID/organ (mean±S.D.) %ID/g (mean±S.D.) To determine the distribution of [186Re]Doxil and [186Re]
[186Re]Doxil [186Re]PEG [186Re] [186Re]PEG PEG liposomes in tumor at 120 h after intravenous
liposomes Doxil liposomes administration, H&E-stained specimens were compared
Blood 1.70±1.20⁎⁎ 0.09±0.07 0.14±0.09 0.01±0.01
with the corresponding autoradiographic images (Fig. 6).
Tumor 0.36±0.33⁎ 0.02±0.01 0.37±0.17 0.02±0.01 Autoradiographically, a higher accumulation of 186 Re
Skin 2.70±1.85⁎ 0.34±0.22 0.1±0.06 0.01±0.01 occurred at the periphery of the tumor than in the center.
Bladder 0.06±0.04 0.03±0.03 0.21±0.13 0.03±0.04 Overlay of the H&E image and autoradiographic images
Stomach 1.30±0.68⁎⁎⁎ 0.11±0.09 0.16±0.09 0.02±0.01 showed that the distribution of radioactivity was, in the
Intestines 0.99±0.27⁎⁎⁎ 0.13±0.07 0.20±0.08 0.02±0.01
Kidney 4.24±1.73 4.90±2.33 5.29±2.06 5.51±2.71
region of expansion, likely a reflection of blood supply.
Spleen 0.83±0.33⁎⁎⁎ 0.16±0.1 5.27±3.39 0.55±0.27 There was decreased accumulation in the tumor center
Liver 4.74±0.52 3.77±1.61 0.56±0.10 0.37±0.14 compared to the periphery. These distributions were similar
Lung 0.14±0.05⁎⁎⁎ 0.03±0.02 0.13±0.03 0.03±0.02 for both [186Re]Doxil and [186Re]PEG liposomes. It has been
Heart 0.03±0.02⁎⁎ 0.01±0.01 0.05±0.03 0.01±0.01 reported that the peripheral tumor capsule of this xenograft
Testis 0.03±0.02⁎⁎ 0.01±0.01 0.02±0.01 0.01±0.004
Bone 1.84±1.08⁎⁎ 0.20±0.11 0.09±0.05 0.01±0.005
had increased vascularity as compared to the central portions
Muscle 1.11±0.95⁎ 0.26±0.19 0.01±0.01 0.003±0.002 of the tumor, as demonstrated with immunohistochemical
Brain 0.01±0.01 0.003±0.002 0.006±0.004 0.002±0.001 stains for endothelial cell markers CD31 and CD34 [24]. The
Urine 11.51±2.19 11.93±1.96 3.14±0.75 1.60±0.18 increased extravasation and accumulation of [186Re]Doxil
Feces 14.80±0.43⁎⁎⁎ 20.75±0.35 11.27±3.71 6.52±0.26 and [186Re]PEG liposomes in the tumor periphery are
Compared with the uptake of [186Re]PEG liposomes in the same organ. expected due to the increased vascularity and integrity of
⁎ Pb.05.
⁎⁎ Pb.01.
blood vessels in this region.
⁎⁎⁎ Pb.001.

in tumor showed a 20-fold increased accumulation for 4. Discussion

[186 Re]Doxil in comparison to [186 Re]PEG liposomes
(Pb.05). The %ID values in the spleen, muscle, skin, testis, Liposomes can be labeled with radioisotopes by trapping
intestines, bone with bone marrow and feces were sig- them within the inner space, intercalated within the double
nificantly higher for [186Re]Doxil than for [186 Re]PEG membrane or connected to the surface of the liposomes [30].
liposomes (Pb.001 for spleen, intestines and feces; Pb.01 for Liposomes have been labeled with β-emitting therapeutic
testis and bone with bone marrow; and Pb.05 for muscle and radionuclides such as 131I, 90Y, 186 Re and 177Lu [21,31,32].
skin). There was no significant difference in %ID values in As the method reported by Hafeli et al. [31] required that
the liver, kidney and urine at 120 h between [186 Re]Doxil 186
Re/188Re complex be labeled by incorporating it into
and [186Re]PEG liposomes. liposomes during liposome manufacturing, this labeling

Fig. 6. Comparison of H&E-stained paraffin sections and autoradiographic images to determine the microdistribution of 186Re in the tumor at 120 h
postadministration of [186Re]Doxil (A) and [186Re]PEG liposomes (B). 186Re is located in the periphery of both tumor specimens. [186Re]Doxil accumulation
was increased in tumor compared to [186Re]PEG liposomes (scale bar, 3 mm).
522 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524

method was clinically impractical. A method of encapsulat- having a clearance time of 28.2 h. The half clearance time of
ing 186 Re/188Re in liposomes with high efficiency, good [186 Re]Doxil is similar to that of 20–30 h for Doxil itself as
stability and convenience was used in the present study [14]. reported previously, suggesting excellent in vivo stability of
Biodistribution and therapy studies have been reported with [186 Re]Doxil [2]. In contrast, [186Re]PEG liposomes had
186
Re neutral liposomes in normal rats [14] and tumor- rapid clearance from the blood and accumulation in the liver.
bearing rats [15,16], respectively. Although labeling of Doxil As the tumors in this study were large (1.5 cm3), they were
with 99mTc and 111In-oxine has been reported [23,33], the characterized by hypovascular areas and increased inter-
labeling method used in this work could directly load stitial pressure, which interfere with passive targeting and
therapeutic 186Re/188 Re into Doxil without prelabeling hence decrease liposome uptake [34]. The slow clearance
modification of the liposomes. rate allowed for the higher bioavailability of [186 Re]Doxil
In this study, the feasibility and characterization of Doxil and thus increased passive targeting of [186Re]Doxil to
labeled with 186 Re were determined for future tumor tumors. Control PEG liposomes were prepared with DSPC,
chemoradionuclide therapy studies. According to the method which has been shown to have a longer circulation time in
reported by Bao et al. [14,23], Doxil was labeled using mice [35]. In spite of similar lipid composition, ammonium
[186Re]BMEDA complex. The [186Re]BMEDA complex sulfate gradient and diameter as Doxil, [186 Re]PEG
was entrapped in Doxil by the ammonium sulfate gradient. liposomes had a very rapid clearance from the blood (Fig.
Control PEG liposomes with similar lipid composition and 3). It has been reported that for liposomes of about 100 nm,
concentration, ammonium sulfate (pH) gradient and particle 9.6 mol% of DSPE-PEG 2000 is required to achieve optimal
size as Doxil were prepared (Table 1) and labeled using the blood circulation and reduced uptake in the liver and spleen
[186Re]BMEDA complex. High labeling efficiencies were [36]. Our control PEG liposomes had only 5 mol% of DSPE-
achieved for [186 Re]Doxil (76.1±8.3%) and [186Re]PEG PEG 2000, and this could be one reason for its faster
liposomes (77.1±8.4%). The in vitro serum stability of clearance. The presence of doxorubicin in crystallized form
[186Re]Doxil was also investigated (Fig. 1). Almost 40% of in Doxil probably renders the liposomes more rigid and
186
Re activity was associated with Doxil at 24 h. The high stable and, hence, 5 mol% of DSPE-PEG 2000 is likely
stability maintained up to 24 h allows for sufficient enough to make it invisible to the reticuloendothelial system
accumulation of [186 Re]Doxil in the tumor. Leakage of the (RES) of the liver and spleen. Further studies on PEG
contents of [186Re]Doxil after 24 h helps release doxorubicin liposomes prepared with an increased mole percentage of
from the liposomes accumulated in the tumor, thus DSPE-PEG 2000 or substitution with DSPE-PEG 5000 [37]
potentially leading to therapeutic effects from both doxor- are required to match the circulation time of Doxil and to
ubicin and 186 Re in the tumor. determine the pharmacokinetics and accumulation in tumor
The in vitro stability achieved with [186 Re]Doxil and for potential therapy.
186
[ Re]PEG liposomes is different from those reported for Molecular imaging has been increasingly applied for drug
186
Re neutral liposomes [14]. This could be potentially due development in preclinical and clinical studies [38] by
to the difference in liposome formulation between Doxil and allowing for the noninvasive assessment of drug efficacy,
neutral liposomes. Since Doxil has a lower amount of pharmacokinetics and distribution in the body. 186 Re is a
cholesterol, 186Re could be released earlier from Doxil/PEG therapeutic radionuclide with a 10% γ emission at 137 keV,
liposomes. Also, 186Re is loaded into an ammonium sulfate which allows for diagnostic imaging and therapy with
gradient occupied by doxorubicin in Doxil and, hence, has [186 Re]Doxil. In this study, imaging of [186 Re]Doxil using
less gradient available for radiolabeling. Another reason planar scintigraphy at various time points and micro-SPECT/
could be that higher initial amounts of 186Re activity per CT at 20 h after intravenous administration depicted
milligram of phospholipid were used for this study compared noninvasively slow blood clearance and low accumulation
with a previous neutral liposome study. Finally, 186Re in the liver. In addition, [186Re]Doxil also accumulated in the
radioactivity also includes stable 185Re nuclide, which can intestine and surrounding tissues, visible from 4 h after
lead to technical challenges in labeling efficiency and administration, and accumulation was stable up to 120 h.
stability compared with the labeling of Doxil using carrier- Activity in the kidney was seen by 46 h for [186Re]Doxil and
free 99mTc or 188Re based on the same BMEDA chemistry. [186 Re]PEG liposomes, suggesting that some of the [186 Re]
The current studies have shown that the high labeling BMEDA released from the metabolized liposomes was
efficiency was not influenced by 185Re carrier; however, in excreted through the kidney. Higher accumulation in the
vitro stability decreased compared with previous reports tumor was seen for [186Re]Doxil in comparison to [186 Re]
using carrier-free 188 Re activity [17–19]. PEG liposomes. The %ID/g values in the blood, tumor, liver,
Although similar in vitro stabilities were observed for spleen, kidney and bowel were determined from planar
[186 Re]Doxil and [186 Re]PEG liposomes, the in vivo scintigraphic images for [186Re]Doxil and [186 Re]PEG
behaviors of [186 Re]Doxil and [186 Re]PEG liposomes had liposomes. Comparison of %ID/g values obtained for the
a profound difference in blood clearance. Pharmacokinetic above organs from imaging and biodistribution at 120 h for
studies of [186Re]Doxil showed prolonged blood retention of [186 Re]Doxil and [186Re]PEG liposomes showed that the
186
Re activity, with 83.17% of the injected [186Re]Doxil values were similar to each other. Thus, the %ID/g values
 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524 523

obtained from imaging at the other time points (4, 20, 46, 70 achievement of a therapeutic effect better than that of
and 96 h) would reflect the trend in the accumulation of doxorubicin and 186Re's individual delivery by liposomes.
radioactivity in the organs after administration and, hence, Further studies are required to evaluate the therapeutic
imaging can be used as a tool for real-time assessment of the efficacy and toxicity of intravenously administered [186Re]
tumor targeting, distribution and pharmacokinetics of Doxil in tumor-bearing rats.
radiolabeled therapeutic liposomes.
Previous biodistribution studies of 186Re neutral lipo-
somes showed high radioactivity in the spleen, liver and 5. Conclusions
kidney at 72 h [14]. The high level of RES organ uptake
with the liposomes is a limitation of liposomal radionuclide Our studies revealed that a high labeling efficiency was
therapy, as high radiation absorbed dose could be delivered achieved for [186 Re]Doxil. [186 Re]Doxil was reasonably
to these organs. In this study, biodistribution at 120 h for stable in 50% FBS and had a long half clearance time in the
[186 Re]Doxil showed high %ID/g values in the spleen and body similar to unlabeled Doxil. The results also demon-
kidney (Table 2). On the basis of results from biodistribu- strated the importance of prolonged circulation time in order
tion and imaging, the kidney would receive a high radiation to achieve improved EPR-based accumulation in tumor. The
absorbed dose from [186Re]BMEDA released from meta- biodistribution, pharmacokinetics and imaging studies of
bolized liposomes and excreted through the kidneys. Thus, [186Re]Doxil in an HNSCC rat xenograft model demon-
the kidney would be the dose-limiting organ of [186 Re] strated good bioavailability, tumor targeting and localization.
Doxil chemoradionuclide therapy. The dose may be reduced Thus, [186Re]Doxil may be used for effective chemoradio-
by using peptides to help remove 186 Re activity from the nuclide therapy with doxorubicin and 186Re's simultaneous
kidney [39,40]. delivery in the same liposome. The therapeutic efficacy of
Active targeting and improved therapeutic efficacy of [186 Re]Doxil will be evaluated in the HNSCC tumor
tumor could be achieved by conjugation of Doxil to a ligand, xenograft model in our future investigations.
peptide or antibodies for immunoliposome drug delivery
[41]. Active targeting could also improve the distribution and Acknowledgments
retention of [186 Re]Doxil in the tumor. The comparison of
[186 Re]Doxil H&E and autoradiography images (Fig. 6) This project was funded by National Institutes of Health
showed a higher accumulation of radioactivity in the tumor supplement grant 5P30CA054174.
periphery, likely a reflection of increased blood supply. Use
of antibodies to target [186Re]Doxil to tumor could help References
achieve homogeneous distribution and retention due to
internalization of the targeted Doxil [42]. Preclinical and [1] Gabizon AA. Pegylated liposomal doxorubicin: metamorphosis of an
clinical studies in breast cancers overexpressing HER2 old drug into a new form of chemotherapy. Cancer Invest 2001;19:
424–36.
showed that HER2 scFv-targeted liposomal doxorubicin [2] Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated
increased the bioavailability and concentration of doxorubi- liposomal doxorubicin: review of animal and human studies. Clin
cin in tumors, which were associated with improved tumor Pharmacokinet 2003;42:419–36.
control [41,43]. By using antibodies to actively target [3] Gabizon AA, Barenholz Y, Bialer M. Prolongation of the circulation
[186 Re]Doxil, the radiation absorbed dose to the tumor time of doxorubicin encapsulated in liposomes containing a poly-
ethylene glycol-derivatized phospholipid: pharmacokinetic studies in
could result in increased DNA damage due to internalization rodents and dogs. Pharm Res 1993;10:703–8.
and improved tumor accumulation. [4] Gabizon AA, Shmeeda H, Zalipsky S. Pros and cons of the liposome
Systemic radionuclide therapy has been effective for platform in cancer drug targeting. J Liposome Res 2006;16:175–83.
hematologic malignancies, but less effective for solid tumors [5] Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer
[44]. The low tumor-to-normal tissue radioactivity ratio could burden: Globocan 2000. Int J Cancer 2001;94:153–6.
[6] Vokes EE, Weichselbaum RR, Lippman SM, Hong WK. Head and
be improved by using liposomes as delivery vehicles. In this neck cancer. N Engl J Med 1993;328:184–94.
study, we showed that a commercial liposome formulation, [7] Kies MS, Bennett CL, Vokes EE. Locally advanced head and neck
Doxil, could be radiolabeled with 186Re for chemoradionu- cancer. Curr Treat Options Oncol 2001;2:7–13.
clide therapy studies. The use of the therapeutic radionuclide [8] Salama JK, Seiwert TY, Vokes EE. Chemoradiotherapy for locally
186
Re allowed for simultaneous imaging of the distribution of advanced head and neck cancer. J Clin Oncol 2007;25:4118–26.
[9] Vokes EE. Interactions of chemotherapy and radiation. Semin Oncol
[186 Re]Doxil and therapy of tumor. Experimental results from 1993;20:70–9.
the pharmacokinetic study revealed a slow clearance rate of [10] Harrington KJ, Lewanski C, Northcote AD, Whittaker J, Peters AM,
[186 Re]Doxil from the blood, which allowed for higher Vile RG, et al. Phase II study of pegylated liposomal doxorubicin
passive targeting to tumor. The biodistribution study at 120 h (Caelyx) as induction chemotherapy for patients with squamous cell
showed a 20-fold increased %ID in the blood and tumor for cancer of the head and neck. Eur J Cancer 2001;37:2015–22.
[11] Harrington KJ, Rowlinson-Busza G, Uster PS, Stewart JS. Pegylated
[186 Re]Doxil in comparison to [186Re]PEG liposomes. These liposome-encapsulated doxorubicin and cisplatin in the treatment of
findings suggest that the increased passive targeting and head and neck xenograft tumours. Cancer Chemother Pharmacol 2000;
concentration of [186Re]Doxil in tumor could result in the 46:10–8.
524 A. Soundararajan et al. / Nuclear Medicine and Biology 36 (2009) 515–524

[12] Harrington KJ, Rowlinson-Busza G, Syrigos KN, Vile RG, Uster PS, [27] Gabizon A, Shiota R, Papahadjopoulos D. Pharmacokinetics and tissue
Peters AM, et al. Pegylated liposome-encapsulated doxorubicin and distribution of doxorubicin encapsulated in stable liposomes with long
cisplatin enhance the effect of radiotherapy in a tumor xenograft circulation times. J Natl Cancer Inst 1989;81:1484–8.
model. Clin Cancer Res 2000;6:4939–49. [28] Stewart JC. Colorimetric determination of phospholipids with
[13] Mitra A, Nan A, Line BR, Ghandehari H. Nanocarriers for nuclear ammonium ferrothiocyanate. Anal Biochem 1980;104:10–4.
imaging and radiotherapy of cancer. Curr Pharm Des 2006;12: [29] Chonn A, Semple SC, Cullis PR. Separation of large unilamellar
4729–49. liposomes from blood components by a spin column procedure:
[14] Bao A, Goins B, Klipper R, Negrete G, Phillips WT. 186Re-liposome towards identifying plasma proteins which mediate liposome clearance
labeling using 186Re-SNS/S complexes: in vitro stability, imaging, and in vivo. Biochim Biophys Acta 1991;1070:215–22.
biodistribution in rats. J Nucl Med 2003;44:1992–9. [30] Phillips WT. Delivery of gamma-imaging agents by liposomes. Adv
[15] Wang SX, Bao A, Herrera SJ, Phillips WT, Goins B, Santoyo C, et al. Drug Deliv Rev 1999;37:13–32.
Intraoperative 186Re-liposome radionuclide therapy in a head and neck [31] Hafeli U, Tiefenauer LX, Schbiger PA, Weder HG. A lipophilic
squamous cell carcinoma xenograft positive surgical margin model. complex with 186Re/188Re incorporated in liposomes suitable for
Clin Cancer Res 2008;14:3975–83. radiotherapy. Int J Rad Appl Instrum B 1991;18:449–54.
[16] Zavaleta CL, Goins BA, Bao A, McManus LM, McMahan CA, [32] McQuarrie S, Mercer J, Syme A, Suresh M, Miller G. Preliminary
Phillips WT. Imaging of 186Re-liposome therapy in ovarian cancer results of nanopharmaceuticals used in the radioimmunotherapy of
xenograft model of peritoneal carcinomatosis. J Drug Target 2008;16: ovarian cancer. J Pharm Pharm Sci 2005;7:29–34.
626–37. [33] Laverman P, Carstens MG, Boerman OC, Dams ET, Oyen WJ, van
[17] Chang YJ, Chang CH, Chang TJ, Yu CY, Chen LC, Jan ML, et al. Rooijen N, et al. Factors affecting the accelerated blood clearance of
Biodistribution, pharmacokinetics and microSPECT/CT imaging of polyethylene glycol-liposomes upon repeated injection. J Pharmacol
188
Re-BMEDA-liposome in a C26 murine colon carcinoma solid Exp Ther 2001;298:607–12.
tumor animal model. Anticancer Res 2007;27:2217–25. [34] Jain RK. Delivery of molecular and cellular medicine to solid tumors.
[18] Chen LC, Chang CH, Yu CY, Chang YJ, Hsu WC, Ho CL, et al. Adv Drug Deliv Rev 2001;46:149–68.
Biodistribution, pharmacokinetics and imaging of (188)Re-BMEDA- [35] Gabizon A, Papahadjopoulos D. Liposome formulations with
labeled pegylated liposomes after intraperitoneal injection in a C26 prolonged circulation time in blood and enhanced uptake by tumors.
colon carcinoma ascites mouse model. Nucl Med Biol 2007;34: Proc Natl Acad Sci U S A 1988;85:6949–53.
415–23. [36] Lee CM, Choi Y, Huh EJ, Lee KY, Song HC, Sun MJ, et al.
[19] Chen LC, Chang CH, Yu CY, Chang YJ, Wu YH, Lee WC, et al. Polyethylene glycol (PEG) modified 99mTc-HMPAO-liposome for
Pharmacokinetics, micro-SPECT/CT imaging and therapeutic efficacy improving blood circulation and biodistribution: the effect of the extent
of (188)Re-DXR-liposome in C26 colon carcinoma ascites mice of PEGylation. Cancer Biother Radiopharm 2005;20:620–8.
model. Nucl Med Biol 2008;35:883–93. [37] Awasthi VD, Garcia D, Goins BA, Phillips WT. Circulation and
[20] Emfietzoglou D, Kostarelos K, Sgouros G. An analytic dosimetry biodistribution profiles of long-circulating PEG-liposomes of various
study for the use of radionuclide–liposome conjugates in internal sizes in rabbits. Int J Pharm 2003;253:121–32.
radiotherapy. J Nucl Med 2001;42:499–504. [38] Willmann JK, van Bruggen N, Dinkelborg LM, Gambhir SS.
[21] Kostarelos K, Emfietzoglou D. Tissue dosimetry of liposome– Molecular imaging in drug development. Nat Rev Drug Discov
radionuclide complexes for internal radiotherapy: toward liposome- 2008;7:591–607.
targeted therapeutic radiopharmaceuticals. Anticancer Res 2000;20: [39] Behr TM, Goldenberg DM, Becker W. Reducing the renal uptake of
3339–45. radiolabeled antibody fragments and peptides for diagnosis and
[22] Zweit J. Radionuclides and carrier molecules for therapy. Phys Med therapy: present status, future prospects and limitations. Eur J Nucl
Biol 1996;41:1905–14. Med 1998;25:201–12.
[23] Bao A, Goins B, Klipper R, Negrete G, Phillips WT. Direct 99mTc [40] Vegt E, van Eerd JE, Eek A, Oyen WJ, Wetzels JF, de Jong M, et al.
labeling of pegylated liposomal doxorubicin (Doxil) for pharmacoki- Reducing renal uptake of radiolabeled peptides using albumin
netic and non-invasive imaging studies. J Pharmacol Exp Ther 2004; fragments. J Nucl Med 2008;49:1506–11.
308:419–25. [41] Park JW, Benz CC, Martin FJ. Future directions of liposome- and
[24] Bao A, Phillips WT, Goins B, McGuff HS, Zheng X, Woolley FR, et al. immunoliposome-based cancer therapeutics. Semin Oncol 2004;31:
Setup and characterization of a human head and neck squamous cell 196–205.
carcinoma xenograft model in nude rats. Otolaryngol Head Neck Surg [42] Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen
2006;135:853–7. UB, et al. Antibody targeting of long-circulating lipidic nanoparticles
[25] Tomayko MM, Reynolds CP. Determination of subcutaneous tumor does not increase tumor localization but does increase internalization in
size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24: animal models. Cancer Res 2006;66:6732–40.
148–54. [43] Laginha KM, Moase EH, Yu N, Huang A, Allen TM. Bioavailability
[26] Ahmed M, Lukyanov AN, Torchilin V, Tournier H, Schneider AN, and therapeutic efficacy of HER2 scFv-targeted liposomal doxorubicin
Goldberg SN. Combined radiofrequency ablation and adjuvant in a murine model of HER2-overexpressing breast cancer. J Drug
liposomal chemotherapy: effect of chemotherapeutic agent, nanopar- Target 2008;16:605–10.
ticle size, and circulation time. J Vasc Interv Radiol 2005;16: [44] DeNardo SJ, Denardo GL. Targeted radionuclide therapy for solid
1365–71. tumors: an overview. Int J Radiat Oncol Biol Phys 2006;66:S89–S95.