Liposomal Cu-64 labeling method using bifunctional chelators: Poly(ethylene glycol) spacer and chelator effects

Article abstract of DOI:10.1021/bc100018n

Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio) propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8, 11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11- carboxymethyl-1,4,8,11-tetraazabicyc

Full text of DOI:10.1021/bc100018n

1206
Bioconjugate Chem. 2010, 21, 1206–1215
Liposomal Cu-64 Labeling Method Using Bifunctional Chelators:
Poly(ethylene glycol) Spacer and Chelator Effects
Jai Woong Seo, Lisa M. Mahakian, Azadeh Kheirolomoom, Hua Zhang, Claude F. Meares,† Riccardo Ferdani,‡
Carolyn J. Anderson,‡ and Katherine W. Ferrara*
Department of Chemistry and Department of Biomedical Engineering, University of California, Davis, California 95616, and
Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110. Received
January 11, 2010; Revised Manuscript Received May 12, 2010
Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-
tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-
carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A-PDEA), were synthesized and conjugated
to long-circulating liposomes (LCLs) via attachment to a maleimide lipid. An in vitro stability assay of 64Cu-
TETA, 64Cu-TETA-PEG2k, and 64Cu-CB-TE2A-PEG2k liposomes showed that more than 86% of the radioactivity
remains associated with the liposomal fraction after 48 h of incubation with mouse serum. The in vivo time
activity curves (TAC) for the three liposomal formulations showed that ∼50% of the radioactivity cleared from
the blood pool in 16-18 h. As expected, the in vivo biodistribution and TAC data obtained at 48 h demonstrate
that the clearance of radioactivity from the liver slows with the incorporation of a poly(ethylene glycol)-2k (PEG2k)
brush. Our data suggest that 64Cu-TETA and 64Cu-CB-TE2A are similarly stable in the blood pool and accumulation
of radioactivity in the liver and spleen is not related to the stability of Cu-64 chelator complex; however, clearance
of Cu-64 from the liver and spleen are faster when injected as 64Cu-TETA-chelated liposomes rather than 64Cu-
CB-TE2A-chelated liposomes.
(t1/2 ) 2.8 days), gallium-67 (t1/2 ) 3.3 days), and technetium-
99m (t1/2 ) 6.01 h) were coordinated with membrane permeable
lipophilic chelators and loaded into the aqueous liposomal core
with a change to a hydrophilic chelator (25, 26, 28, 30). As an
insertion method, radiolabeled lipids, 3-[18F]fluoro-1,2-dipalmi-
toylglycerol ([18F]-FDP) or [18F]-labeled amphiphilic lipids, were
incorporated into the liposomal bilayer (31, 33). Surface chelation
methods, the most convenient method, were also developed with
technetium-99m or copper-64 (t1/2 ) 12.7 h) by covalently
attaching the chelator on the surface of the liposome (27, 36)
or conjugating 18F-labeled PEG-acetylene with an azide group
on the liposome via a “click” reaction (37).
The previous labeling approaches were focused on obtaining
a stable conjugation of a radionuclide and liposome to quantify
the liposomal activity in target tissues over days to weeks
without the radionuclide undergoing metabolism or becoming
decomplexed. We previously developed a liposomal Cu-64
labeling method using 6-[p-(bromoacetamido)benzyl]-1,4,8,11-
tetraazacyclotetradecane-N,N′,N′′,N′′′-tetraacetic acid (BAT)
conjugated lipid (6-BAT-lipid) and successfully measured the
liposomal circulation time in blood over 48 h (36). However,
liposomes with high molar concentrations of peptide have shown
a lower radiolabeling yield. CB-TE2A, which forms a highly
stable complex with Cu-64, has not previously been explored
in a liposomal study using a postlabeling method because the
chelation of Cu-64 should be performed at a temperature above
80 °C, which may affect liposomal stability due to the intrinsic
transition temperature of the liposome (38, 39).
INTRODUCTION
Liposomes, spherical vehicles composed of a lipid bilayer
with an aqueous interior, have been investigated for more than
40 years as a promising drug carrier (1-3). When coated by
poly(ethylene glycol), liposomal circulation time in blood
increased with a decrease in reticuloendothelial system (RES)
uptake (4-6). Currently, 90-100-nm-diameter liposomes en-
trapping anticancer or antifungal drugs are approved and used
for passively targeting the relatively leaky vasculature of
tumors (7, 8). Advanced liposomal technology exploits
peptide (9, 10), antibody (11, 12), and small molecule
ligands (13, 14) by attaching these ligands on the surface of
liposomes to maximize the retention at the target site. In
addition, triggered drug release by ultrasound (15, 16) or a
laser (17, 18) and in a low pH environment (19) has been
explored for local delivery.
Noninvasive imaging techniques using SPECT and PET
have been developed to evaluate the pharmacokinetics and
biodistribution of liposomes in vivo (20-22). Previous
studies have utilized liposomes with radionuclides such as
technetium-99m (23-27), gallium-67 (28), and indium-111
(29, 30) for single photon emission computed tomography
(SPECT) and fluorine-18 (31-33), iodine-124 (34), oxygen-
15 (35), and copper-64 (36) for positron emission tomography
(PET), where radionuclides were applied with three distinct
schemes involving entrapment, insertion, and chelation. In
the entrapment method, radionuclides such as indium-111
Recent studies of targeted nanoparticles combine antibodies,
peptides, small molecules, and polymers on the surface to
optimize and personalize targeting (9-14). For this reason, it
is important to optimize radiochemistry techniques for specific
surface architectures, and therefore, alternative postlabeling
methods and CB-TE2A are explored. Herein, we developed and
evaluated a Cu-64 labeling method using a maleimide lipid and
* To whom correspondence should be addressed. Prof. Katherine
W. Ferrara, Dept. of Biomedical Engineering, 451 East Health Sciences
Drive, University of California, Davis, Davis, CA 95616. Phone 530-
754-9436, fax (530)754-5739, e-mail: kwferrara@ucdavis.edu.
† Department of Biomedical Engineering, University of California,
Davis.
‡ Washington University School of Medicine.
10.1021/bc100018n  2010 American Chemical Society
Published on Web 06/22/2010

Liposomal Cu-64 Labeling Using Bifunctional Chelators
Bioconjugate Chem., Vol. 21, No. 7, 2010 1207
bifunctional chelator (BFC). Stability in vitro and in vivo is
then compared for liposomes labeled with TETA-PDP and CB-
TE2A-PDEA and with varied spacer lengths between the lipid
and the chelator.1
distilled water; gradient, 0-15 min (20-25% A), 15-30 min
(25-90% A); retention time, 9.3 ( 0.35 min). The collected
product fraction was lyophilized and 4 obtained as a white
powder (19 mg, 74%). 1H NMR (500 MHz, D2O): 8.58 (d, J )
5.0 Hz, 1H), 8.23 (t, J ) 8.5 Hz, 1H), 8.13 (d, J ) 8.5 Hz,
1H), 7.64 (t, J ) 6.5 Hz, 1H), 4.22 (d, J ) 15.5 Hz, 1H), 4.02
(d, J ) 16.0 Hz, 1H), 3.76-3.62 (m, 3H), 3.62-3.51 (m, 2H),
3.50-3.34 (m, 4H), 3.25-2.90 (m, 15H), 2.75 (bd, J ) 13.5
Hz, 1H), 2.75 (dd, J ) 13.5, 2.0 Hz, 1H), 2.45-2.31 (m, 2H),
1.75 (t, J ) 16.5 Hz, 2H); MS (ESI) m/z 511.5 (M+H+, 100).
HPLC Analysis of Cu-64 Chelation with CB-TE2A-PDEA.
A few microliters of 64CuCl2 were buffered with 0.1 M
ammonium citrate (100 µL). CB-TE2A-PDEA (1 mM, 2 µL)
dissolved in ammonium citrate buffer (0.1 M, pH 5.5) was added
into a buffered 64CuCl2 solution, and the mixture was incubated
at 85 °C. Samples for HPLC analysis were acquired at 0, 30,
and 60 min and kept in an ice bath until they were injected into
the HPLC. Freshly prepared TCEP (10 mM, 2 µL) in PBS was
then added, and the mixture was held at room temperature for
10 min and applied to an HPLC column (Jupiter, C12, 250 ×
4.6 mm, flow rate, 1 mL/min; eluent, A - 0.5% TFA in deionized
water, B - 0.5% TFA in acetonitrile, 0-10 min (10-20% B),
10-20 min (20-100% B), 20-24 min (100% B), 24-25 min
(100-10% B)).
Preparation of Maleimide Liposomes. Commercially avail-
able lipids (10 mg of HSPC, cholesterol, DSPE-PEG2k-OMe,
and 1 mol % DSPE-PEG2k-maleimde or DSPE-maleimide) in
chloroform were mixed in a test tube. Chloroform was
evaporated by a gentle stream of nitrogen with a vortex. After
drying overnight in a lyophilizer, lipids were suspended in PBS
(1×). The lipid solution was incubated at 60 °C for 5 min, drawn
into the 1 mL syringe, and extruded at 60 °C by a mini extruder
unit with 100 nm filters. Immediately after extrusion, the size
was measured using a NICOMP 380 ZLS (Particle Sizing
Systems, CA), and liposomes were labeled with Cu-64 labeled
bifunctional chelators. The average liposomal diameter was
123.6 ( 13.0 nm.
Labeling of Maleimide Liposomes with BFCs. All labeling
procedures were controlled under a protocol of the University
of California, Davis. 64CuCl2 (0.55-5 mCi, [18.5-185 MBq])
was buffered with 0.1 M ammonium citrate (100 µL). TETA-
PDP or CB-TE2A-PDEA (1 mM, 10-2 µL) in an ammonium
citrate buffer (0.1 M, pH 5.5) was added into a 64CuCl2 solution,
which was incubated for 40 min at 30 °C or 1.5 h at 85 °C,
respectively. The reaction progress was monitored by radio TLC
(silica backed plate, eluent 10% (w/w) ammonium acetate and
methanol (1:1, v/v)) or radio HPLC. After the addition of 10×
excess of 0.1 M Tris(2-carboxylethyl)phosphine hydrochloride
(TCEP·HCl) dissolved in PBS, the solution was incubated for
5 min at room temperature. Freshly prepared maleimide
liposomes (10, 5, 2.5, and 1 mg) in PBS (0.4 mL) were added
to the reduced bifunctional chelator mixture. The pH was
adjusted with 5.6% (v) ammonium hydroxide solution, and the
liposomal mixture was incubated for 40 min at 30 °C. After
cooling of the liposomal mixture, unreacted maleimide on the
surface of the liposomes was quenched by an excess (maleimide/
ethanethiol ) 1:10, mol/mol) of freshly prepared 0.1 M
ethanethiol solution by incubating for 10 min. Lastly, nonspecific
binding of Cu-64 was removed by 0.1 M EDTA (20 µL)
incubation for 5-10 min. Liposomes were purified by size
exclusion chromatography with PBS.
Materials. HSPC, DSPE-PEG2k-maleimide, cholesterol,
DSPE-PEG2k-OMe, were purchased from Avanti Polar Lipids
(Alabaster, AL), and DSPE-maleimide was purchased from NOF
America Corporation (White Plains, NY). Sulfo-LC-SPDP
(sulfosuccinimidyl 6-[3-(2-pyridyldithio)-propionamido]hex-
anoate) was purchased from Thermo Scientific (Rockford, IL).
S-(2-Aminoethyl)dithio-2-pyridine hydrochloride was purchased
from Toronto Research Chemicals (North York, ON). Solvents
and other agents were all of analytical purity and purchased
from Sigma-Aldrich (Milwaukee, WI) and VWR (Brisbane,
CA). 64CuCl2 was purchased from Isotrace (St. Louis, MO)
under a protocol controlled by the University of California,
Davis. PBS was purchased from Invitrogen Corporation (Carls-
bad, CA). Mouse serum was purchased from Innovative research
(Novi, MI).
EXPERIMENTAL SECTION
Synthesis of TETA-PDP (2). (6-(6-(3-(2-Pyridyldithio)pro-
pionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetrade-
cane-1,4,8,11-tetraacetic acid (TETA-PDP, 2) was synthesized
from aminobenzyl-TETA (1) based on a previously reported
method (40, 41). To a solution of 1 (18 mg, 34 µmol) in DI
water (2 mL, pH 7) was added sulfosuccinimidyl-6-[3′-(2-
pyridyldithio)-propionamido]hexanoate (22 mg, 42 µmol). The
mixture was stirred at room temperature for 4 h. Reaction
progress was monitored by a semipreparative HPLC (column,
Jupiter 10 µ proteo, 250 × 10 mm; flow rate, 3 mL/min; eluent,
A - MeOH, B - 0.05% TFA in triple-distilled water; gradient,
0-15 min (40-80% A), 15-30 min (80-100% A)) and
retention time was 11 min. Coupled product 2 was isolated by
preparative HPLC (column, Jupiter 10 µ, 250 × 21.2 mm; flow
rate, 10 mL/min; eluent, A - MeOH, B - 0.05% TFA in triple-
distilled water; gradient, 0-15 min (40-80% A), 1-30 min
(80-100% A); retention time, 13.3 ( 0.36 min) and lyophilized
as a white solid (25 mg, 87%). 1H NMR (500 MHz, D2O): 8.48
(d, J ) 5.5 Hz, 1H), 8.12 (t, J ) 8.0 Hz, 1H), 7.91 (d, J ) 8.5
Hz, 1H), 7.55 (t, J ) 6.0 Hz, 1H), 7.36 (d, J ) 7.5 Hz, 2H),
7.30 (d, J ) 8.0 Hz, 2H), 3.35-3.65 (m, 12H), 0.2.95-3.32
(m, 16H), 2.61 (t, J ) 6.5 Hz, 2H), 2.55-2.45 (m, 3H), 2.08
(bs, 1H), 1.78 (bs, 1H), 1.73 (m, 2H), 1.54 (m, 2H), 1.39 (m,
2H); MS (ESI) m/z 848.4 (M+H+).
Synthesis of CB-TE2A-PDEA (4). 4-(2-(2-Pyridyldithio-
ethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo-
(6.6.2)hexadecane (CB-TE2A-PDEA, 4) was synthesized from
CB-TE2A (3) as in refs 42, 43. To a solution of 3 (68 mg, 0.1
mmol) in DMF (4 mL) was added N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride (EDCI ·HCl, 9.6 mg, 50
µmol), N-hydroxybenzotriazole (HOBt, 8.1 mg, 60 µmol), and
N,N-diisopropylethylamine (DIPEA, 52 µL, 0.3 mmol). After
stirring for 30 min at room temperature, S-(2-aminoethyl)dithio-
2-pyridine hydrochloride (11 mg, 50 µmol) was added and the
reaction mixture was stirred overnight. The solvent was removed
under high vacuum, and residual oil was injected into the
preparative HPLC after dilution of oil with a 0.05% TFA
solution (column, Jupiter 10 µ, 250 × 21.2 mm; flow rate, 10
mL/min; eluent, A - acetonitrile, B - 0.05% TFA in triple-
In Vitro Stability Test. 64Cu-labeled liposomes (64Cu-TETA,
64Cu-TETA-PEG2k, and 64Cu-CB-TE2A-PEG2k liposomes, 1
mg, 10.1-12.28 MBq) were added to 0.5 mL of mouse serum
and PBS, and the mixture was incubated for 48 h at 37 °C.
After cooling, a portion of the liposomal serum mixture (100
µL) was hand-loaded onto a preconditioned Sephacryl-300HR
1Abbreviations: HSPC, hydrogenated soy L-R-phosphatidylcholine;
DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; DSPE-PEG2k-
OMe, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-
(polyethylene glycol)-2000]; MI, maleimide; HOBt, N-hydroxybenzo-
triazole; EDCI, (3-(dimethylamino)propyl)ethyl carbodiimide; DIPEA,
diisopropylethylamine.

1208 Bioconjugate Chem., Vol. 21, No. 7, 2010
Seo et al.
Scheme 1. Synthesis of Bifunctional Chelators (BFCs) Bearing TETA and CB-TE2Aa
a Reagents: (a) Sulfo-LC-SPDP (sulfosuccinimidyl-6-[3′-(2-pyridyldithio)propionamido]hexanoate), DI water, rt, 4 h. (b) S-(2-Aminoethyl)dithio-
2-pyridine hydrochloride, HOBt, EDCI, DIPEA, DMF, rt, 6 h.
(GE Healthcare, NJ) packed column (15 mm i.d., 250 mm
height) and separated with PBS at 2 psi. Fractions (1.5 mL/
fraction) were collected into test tubes, and the radioactivity
was measured with the 1470 Automatic Gamma Counter
(Perkin-Elmer Life Sciences, MA). After the radioactive decay,
the absorbance of all fractions was measured at 280 nm. The
radio TLC assay was run on aluminum-backed silica gel sheets
(silica gel 60 F254, EMD, NJ), developed with chloroform/
methanol/H2O (50:40:6, v/v/v) and recorded by a radio-TLC
Imaging Scanner (Bioscan, NW).
Biodistribution Study. All animal studies were conducted
under a protocol approved by the University of California, Davis,
Animal Use and Care Committee. A total of 13 animals (female
FVB mice, 12-19 weeks, 25-30 g, Charles River, MA) were
examined over the course of this study. For each image, 4 or 5
mice per group were anesthetized with 3.5% isoflurane and
maintained at 2.0-2.5%. 64Cu-TETA liposomes (38 ( 0.7 mg/
kg, 313 ( 19 µCi [11.58 ( 0.70 MBq]/animal), 64Cu-TETA-
PEG2k liposomes (43 ( 6.7 mg/kg, 332 ( 55 µCi [12.28 (
2.03 MBq]/animal), and 64Cu-CB-TE2A-PEG2k liposomes (34
( 1.6 mg/kg, 272 ( 9.2 µCi [10.06 ( 0.34 MBq]/animal) in
PBS (pH 7.4) were administered to the mice on a PET scanner
bed by catheterized tail vein injection. After scanning over 48 h,
the mice were euthanized by cervical dislocation, and organs
of interest were harvested and weighed. Radioactivity was
measured using a 1470 Automatic Gamma Counter.
PET Scans and Time-Activity Curves (TAC). Two mice
were placed on one bed, and each set of PET scans was
conducted with the microPET Focus (Concorde Microsystems,
Inc., TN) over 30 min. Mice were scanned at an initial time
point (t ) 0), and 6, 18, 28, and 48 h. Filtered backprojection
(FBP) reconstruction was obtained with ASIPro software (CTI
Molecular Imaging). TACs were obtained with region-of-interest
(ROI) analysis using ASIPro software and expressed as the
percentage of injected dose per cubic centimeter (%ID/cc).
Statistical Analysis. Prism (GraphPad Software Inc., San
Diego, CA) was used for statistical analysis. One-phase
exponential blood clearance curves were generated via a curve
fit. The data analysis was performed using one-way analysis of
variance (ANOVA).
from trace metals, aminobenzyl TETA 1 was coupled with
sulfosuccinimidyl-6-[3′-(2-pyridyldithio)propionamido]hex-
anoate in distilled water at pH 7 (Scheme 1). The product
was isolated by preparative HPLC with retention time on
average 13.3 min, and overall synthesis yield was 10%. The
final product was validated by 1H NMR spectral analysis and
electrospray ionization-mass spectrometry in a positive
mode. A single charged peak (20%) at 848 and a double
charged peak (100%) at 425 were detected.
Synthesis of CB-TE2A-PDEA (4). A half equivalent of S-(2-
aminoethyl)dithio-2-pyridine hydrochloride and CB-TE2A was
treated with HOBt, EDCI, and DIPEA in DMF to obtain the
carboxylic acid-coupled product (Scheme 1). DMF was removed
under high vacuum to achieve a consistent retention time of
9.3 min on preparative HPLC. Lyophilization of the product
fractions yielded 74% CB-TE2A-PDEA. The product was also
validated by 1H NMR spectral analysis and electrospray
ionization-mass spectrometry in a positive mode. A single
charged peak was detected at 511 (100%).
Radio HPLC Analysis of Cu-64 Incorporation into CB-
TE2A-PDEA and Formation of Active Sulfhydryl Group.
Incorporation of Cu-64 with TETA is performed at room
temperature within 1 h. On the other hand, CB-TE2A requires
heating (>80 °C) for incorporation of Cu-64. First, we
evaluated the stability of CB-TE2A-PDEA under the chela-
tion conditions at 85 °C. Freshly prepared 2 nmol of 1 mM
CB-TE2A-PDEA 4 in ammonium acetate buffer (pH 5.5, 100
µL) was incubated with a few hundred µCi of Cu-64 at 85
°C. The reaction progress and decomposition of Cu-64
incorporated BFC were monitored by HPLC analysis. After
the addition of 64CuCl2 into the CB-TE2A-PDEA solution,
the radiochromatogram showed that only Cu-64 peak was
eluted at 3 min (a, Figure 2). After incubation for 30 and 60
min, 69% and 96% of the radioactivity was observed with a
retention time of 15 min, respectively (b and c, Figure 2),
which indicated that Cu-64 was completely incorporated into
CB-TE2A-PDEA without decomposition. After the Cu-64
chelation, 64Cu-CB-TE2A-PDEA solution was cooled to room
temperature for 10 min, and the addition of a 10× excess
amount of TCEP (10 mM, 2 µL) as a reducing reagent
selectively cleaved the disulfide bond (44). Incubation for
10 min converted all Cu-64-incorporated-CB-TE2A-PDEA
into the peak at 8 min. Thus, the pyridyldithiol 6 was rapidly
reduced to the sulfhydryl group 7 (d, Figure 2) with more
than 99% yield, which was confirmed by the conversion of
retention time of 6 to 7.
RESULTS
Synthesis of TETA-PDP (2). Aminobenzyl TETA 1,
precursor of TETA-PDP, was achieved in 5 steps from
4-nitrobenzyl bromide as previously reported (40, 41). Critical
steps in determining the yield were cyclization, where the
resulting yield was 40% with N,N-bis(2-aminoethyl)-1.3-
propanediamine, and alkylation, where the resulting yield was
41% with benzylbromoacetate. To reduce the contamination
BFC Conjugation Study with Maleimide Liposome. Lip-
osomes (average diameter: 119 ( 12 nm) were prepared from
HSPC, DSPE-PEG2k-OMe, cholesterol, and DSPE-maleimide

Liposomal Cu-64 Labeling Using Bifunctional Chelators
Bioconjugate Chem., Vol. 21, No. 7, 2010 1209
Figure 3. (a) Structure of maleimide lipids with and without PEG. (b)
Formulations of long circulating liposomes and modified formulations.
Two different 1 mol % maleimide lipids were included.
liposomes, and 20 ( 4%, 47 ( 17%, 92 ( 3%, and 94 ( 3%
(n ) 4) for 1 mol % maleimide-PEG2k-liposomes (Figure 5).
The radiochemical purity checked by the separated liposomal
fraction was more than 99% on radio TLC. The specific activity
of Cu-64 labeled liposomes in this in vivo study was 20.8 (
1.6 mCi/µmol maleimide lipid.
Figure 1. General scheme of liposomal postlabeling with (BFC).
In Vitro Stability Test. The in vitro stability of BFC
conjugated maleimide liposomes was assessed with 64Cu-TETA,
64Cu-TETA-PEG2k, and 64Cu-CB-TE2A-PEG2k liposomes
(each 1 mg) in PBS/mouse serum (1/1, v/v) after 48 h incubation
at 37 °C. After size exclusion chromatography (n ) 3), we
obtained 84 ( 2% of the total radioactivity in 50 fractions (75
mL) from all liposomes. Liposomal fractions eluted from
fraction 8 to 15 contain 89% (64Cu-TETA liposomes), 86%
(64Cu-TETA-PEG2k liposomes), and 86% (64Cu-CB-TE2A-
PEG2k liposomes) of total eluted radioactivity (Figure 6).
Radiochemical purity, monitored by radio TLC, of each
liposomal fraction was more than 98%.
In Vivo PET Images and Time-Activity Curves (TAC). In
vivo imaging with 64Cu-BFC-labeled liposomes from 30 min
scans at 0, 6, 18, and 28 h after intravenous injection
demonstrated long circulation of the 64Cu-TETA liposomes (1.0
mg, 313 ( 19 µCi [11.58 ( 0.70 MBq]), 64Cu-TETA-PEG2k
liposomes (1.2 mg, 332 ( 56 µCi [12.28 ( 2.03 MBq]), and
64Cu-CB-TE2A-PEG2k liposomes (1.0 mg, 272 ( 9.2 µCi
[10.06 ( 0.34 MBq]) (Figure 7). Liposomal activity was high
in the blood pool from 0 to 6 h and slowly cleared out through
the reticuloendothelial system. Time activity curves (TAC)
corresponding to blood, liver, and spleen are shown in Figure
8. For 64Cu-TETA liposomes, radioactivity in the blood pool
decreased to 41 ( 1.2%, 34 ( 1.6%, 20 ( 0.5%, 13 ( 1.0%,
and 8.4 ( 0.6%ID/cc at 30 min and 6, 18, 28, and 48 h,
respectively (Figure 8b). At the same time points, with 64Cu-
TETA-PEG2k liposomes, the radioactivity in the blood pool
was 41 ( 3.8%, 31 ( 3.3%, 20 ( 2.0%, 13 ( 2.3%, and 7.6
( 0.6%ID/cc, for 64Cu-CB-TE2A-PEG2k liposomes, 39 (
1.1%, 28 ( 2.3%, 18 ( 2.1%, 11 ( 1.0%, and 6.4 ( 1.6%ID/
cc at 30 min and 6, 18, 28, and 48 h, respectively (Figure 8b).
Exponential curve-fitting of three liposomal formulations showed
one-phase exponential clearance from the blood pool. TAC data
obtained from the blood pool was simulated with one-phase
exponential decay as shown in Table 2.
Figure 2. HPLC radio chromatogram: (a) immediately after mixing
64CuCl2 and CB-TE2A-PDEA at room temperature; (b) after 30 min
incubation at 85 °C; (c) after 60 min incubation at 85 °C; (d) after
addition of 10 equiv of tris-(2-carboxyethyl)phosphine (TCEP) at room
temperature.
(molar ratio 55/5/39/1) and HSPC, DSPE-PEG2k-OMe, cho-
lesterol, and DSPE-PEG2k-maleimide (molar ration 56/4/39/
1) (Figure 3) as described in the experimental procedure. The
incorporation of Cu-64 (0.2-1 mCi [7.4-37 MBq]) into TETA-
PDP 2 (2 nmol) at 30 °C was successful (>99%), which was
confirmed by the Rf value of radio TLC (64CuCl2, Rf 0; 64Cu-
TETA-PDP, Rf 0.45). Treatment by TCEP (10 equiv/TETA-
PDP), which reduces a disulfide over a wide pH range (45),
converted pyridyldithiol 8 to the sulfhydryl compound 9 with
more than 99% yield within 10 min (data not shown but similar
to Figure 2). To increase the nucleophilicity of the thiols, the
pH was adjusted to 7.0-7.5. The conjugation yield of 64Cu-
TETA-thiol 9 was assessed with 0.5, 1.25, and 2.5 mg of
maleimide liposomes which have 7, 17, 35, and 70 nmol of
maleimide on the surface of liposome, respectively. The
corresponding liposomal conjugation yields were 16 ( 2%, 43
( 14%, 91 ( 3%, and 93 ( 3% (n ) 4) for 1 mol % maleimide
For the three liposomal preparations, estimated radioactivity
in the liver was 10.5 ( 0.8%, 13.3 ( 0.6%, 12.1 ( 0.9%, 8.7
( 0.1%, and 6.7 ( 0.2%ID/cc (64Cu-TETA liposomes), 8.1 (

1210 Bioconjugate Chem., Vol. 21, No. 7, 2010
Seo et al.
Figure 4. Liposomal labeling model with 64Cu-TETA-PDP and two maleimide liposomes.
injected mice, radioactivity in the liver (6.88 ( 0.96%ID/g) and
spleen (12.26 ( 0.70%ID/g) was significantly lower than that
of the liver (15.2 ( 1.23%ID/g, 22.9 ( 4.42%ID/g) and spleen
(20.3 ( 2.49%ID/g, 30.4 ( 5.25%ID/g) of 64Cu-TETA-PEG2k
and 64Cu-CB-TE2A-PEG2k liposome-injected mice, respec-
tively. Other organs showed a similar biodistribution for the
three liposomal formulations (Table 1).
DISCUSSION
The first aim of this study was to develop a liposomal
conjugation method using a Cu-64 and thiol-reactive bifunctional
chelator. Since the conjugation of peptides on the liposomal
surface has utilized the reaction of thiol with maleimide within
5 to 60 min at 25 °C (46-48), liposomal conjugation with a
thiol-activable Cu-64-incorporated bifunctional chelator was
considered to be a promising approach. On the basis of this
aim, we proposed the scheme outlined in Figure 1.
The incorporation of Cu-64 to TETA-PDP was performed
under mild conditions (30 °C) within an hour, and the reaction
progress could easily be confirmed by radio TLC (Rf 0.45
with 10% ammonium acetate and methanol (1:1, v/v)).
However, the reaction progress of 4 could not be monitored
by radio-TLC due to the similar retention factor (both Rf )
0) of 64CuCl (5) and 64Cu-CB-TE2A-PDEA (6). In addition,
the stability of pyridine dithiol in CB-TE2A-PDEA (4) during
chelation at 85 °C was a concern. Accordingly, the reaction
progress was assessed by HPLC analysis (Figure 2). Radio
HPLC analysis demonstrated that the chelation of Cu-64 into
CB-TE2A-PDEA (4) was completed within 1 h, and the
conversion of 6 to thiol (7) succeeded without decomplex-
ation of Cu-64 from the chelators or decomposition of 6 and
7 (Figure 2).
Figure 5. Labeling yield (n ) 4) of maleimide (1 mol %) liposomes
with various molar ratios of Cu-64-incorporated TETA-BFC at pH 7
(black bar, 1 mol % maleimide liposomes; white bar, 1 mol %
maleimide-PEG2k liposomes).
1.5%, 14.3 ( 1.0%, 15.1 ( 0.9%, 13.7 ( 1.3%, and 11.0 (
0.8%ID/cc (64Cu-TETA-PEG2k liposomes), and 7.4 ( 0.9%,
13.5 ( 2.0%, 16.5 ( 2.2%, 15.4 ( 2.0%, and 15.7 ( 1.7%ID/
cc (64Cu-CB-TE2A-PEG2k liposomes) at 30 min, and 6, 18,
28, and 48 h, respectively (Figure 8c). Estimated radioactivity
in the spleen was 6.0 ( 1.7%, 8.1 ( 4.1%, 8.1 ( 1.0%, 4.8 (
1.3%, and 4.2 ( 0.9%ID/cc (64Cu-TETA liposomes), 7.5 (
0.8%, 9.6 ( 1.2%, 10.2 ( 2.1%, 9.4 ( 2.4%, and 8.3 (
2.0%ID/cc (64Cu-TETA-PEG2k-liposomes), and 6.9 ( 0.6%,
10.8 ( 1.3%, 14.8 ( 3.6%, 13.2 ( 4.5%, and 10.4 ( 2.7%ID/
cc (64Cu-CB-TE2A-PEG2k liposomes) at 30 min, 6, 18, 28, and
48 h, respectively (Figure 8d).
Biodistribution. The biodistribution at 48 h confirmed the
TAC estimates (Table 1). There was no significant difference
in blood radioactivity (8.71 ( 0.38%, 8.35 ( 1.10%, and 6.85
( 1.66%ID/g) at 48 h after administration of 64Cu-TETA
liposomes, 64Cu-TETA-PEG2k liposomes, and 64Cu-CB-TE2A-
PEG2k liposomes, respectively. For 64Cu-TETA liposome-
Next, we moved forward to the optimization of labeling yield,
as outlined in Figure 4. For this study, 1 mol % maleimide and

Liposomal Cu-64 Labeling Using Bifunctional Chelators
Bioconjugate Chem., Vol. 21, No. 7, 2010 1211
Figure 7. Micro-PET images (left, coronal; right, sagittal) acquired
after injection of 64Cu-TETA liposomes (a1-4); 64Cu-TETA-PEG2k
liposomes (b1-4); and 64Cu-CB-TE2A-PEG2k liposomes (c1-4)
immediately after injection (a1-c1); 6 h later (a2-c2); 18 h later
(a3-c3); and 28 h later (a4-c4). All images were acquired after
reconstruction by filtered backprojection.
mCi/µmol maleimide lipids. Preclinical studies of Doxil (long
circulating liposomes) typically use 2.5-20 mg lipids/kg
mouse for the treatment of tumor-bearing mice (7). In our
model, approximately 40 mg/kg lipids were administered for
the Cu-64 labeled liposomal study, and a high signal-to-noise
ratio was achieved. The liposome dose can be reduced if
imaging with Cu-64 labeled liposomes shows tumor ac-
cumulation at a lower liposome dose, and particularly if the
Cu-64 has a high effective specific activity (>6000 mCi/µmol
Cu-64) (49).
Previously, 99mTc-BMEDA encapsulated liposomes (50) and
99mTc-HYNIC surface-labeled liposomes (26) have been con-
sidered stable labeling methods for liposomal SPECT studies.
For liposomal PET studies, our prior labeling method showed
sufficient stability to represent liposomal behavior in vivo (36).
In this connection, the second step of the study was to evaluate
the liposomal stability in vitro and in vivo after the conjugation
of different bifunctional chelators (6 and 8). The stability of 1
mol % maleimide-PEG2k liposomes was compared after the
conjugation with 64Cu-TETA-PDP and 64Cu-CB-TE2A-PDEA
to evaluate the effect of the chelator. Maleimide and maleimide-
PEG2k liposomes were compared after labeling with 64Cu-
TETA-PDP to determine whether the conjugated 64Cu-TETA-
PDP is stable under a PEG2k brush or the end of a PEG2k
lipopolymer. To answer those questions, the stability of three
liposomal formulations (64Cu-TETA, 64Cu-TETA-PEG2k, and
64Cu-CB-TE2A-PEG2k liposomes) was examined in vitro and
vivo.
In vitro assays performed with 64Cu-TETA, 64Cu-TETA-
PEG2k, and 64Cu-CB-TE2A-PEG2k liposomes showed that
89%, 86%, and 86% radioactivity are still associated with the
liposomal fraction, respectively, at 48 h (Figure 6). This result
was similar to that obtained in our previous study (in which
88% of the radioactivity was in the liposomal fraction) (36).
The radiochemical purity of each liposomal fraction determined
with radio TLC was more than 98%.
Figure 6. In vitro assay of Cu-64-labeled maleimide liposomes.
Chromatograms were obtained after 48 h incubation in mouse serum
and PBS (1:1, v/v) bearing (a) 64Cu-TETA liposomes; (b) 64Cu-
TETA- PEG2k liposomes; and (c) 64Cu-CB-TE2A-PEG2k liposomes.
Red line represents radioactivity of liposomes detected by a gamma
counter and blue line represents serum proteins detected by UV-
absorbance at 280 nm.
1 mol % maleimide-PEG2k liposomes were employed to
compare the labeling efficiency and to determine whether the
PEG2k brush decreases the conjugation (Figure 3). In order to
maintain 5 mol % PEG2k on the surface of the liposomes, which
facilitates long circulation of liposomes in the blood pool, either
5 mol % DSPE-PEG2k-OMe or a combination of 4 mol %
DSPE-PEG2k-OMe and 1 mol % DSPE-PEG2k maleimide were
incorporated within our formulations (Figure 3). The labeling
study (measured in four molar ratios of maleimide lipid versus
TETA-PDP (2)) showed that at least an 8-fold lower molar
concentration of 2 should be incubated with both liposomes to
obtain greater than 90% yield (pH 7, 40 min incubation) (Figure
5). The BFC is fully accessible with similar labeling efficiency
whether the maleimide sits under the PEG2k-brush or at the
end of the PEG2k lipid.
In general, more than 6000 mCi/µmol maleimide lipids
could be obtained from Cu-64 generated from a Ni-64 target
(49) under the assumption of 100% chelation of Cu-64 with
the BFC and 100% conjugation of 64Cu-BFC to the maleimide
lipid. However, due to the excess BFC (10 times more than
Cu-64) and maleimide (7 times more than 64Cu-BFC), the
specific activity calculated from maleimide lipids was 20.8
A normal rat study performed by Bao et al. using 99mTc-
BMEDA encapsulated liposomes reported a two-phase blood
clearance of liposomes (50). Forty percent of activity
remained in the blood pool at 18 h based on the decay
function in their study (y ) 36.4 × e-0.315×t + 63.7 × e-0.0264×t
rats studied by Laverman et al. using 99mTc-HYNIC surface-
,
y ) % activity in blood, t ) time (h)). S. aureus infected

1212 Bioconjugate Chem., Vol. 21, No. 7, 2010
Seo et al.
Figure 8. (a) Whole body activity clearance (decay-corrected). Time activity curves (TACs, %ID/cc) of heart (b), liver (c), and spleen (d) (n )
4-5) obtained from microPET by drawing regions of interest (ROI) after the injection of 64Cu-TETA liposomes (orange square), 64Cu-TETA-
PEG2K liposomes (green diamond), and 64Cu-CB-TE2A-PEG2k liposomes (blue round). ANOVA followed by Tukey’s multiple comparison test
was performed (error bars, mean ( STD; n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001).
Table 1. Biodistribution of 64Cu-TETA Liposomes (n ) 4), 64Cu-TETA-PEG2K Liposomes (n ) 5), and 64Cu-CB-TE2A-PEG2k Liposomes (n )
4) in Mice at 48 h after Injectiona
64Cu-TETA liposomes (n ) 4)
64Cu-TETA-PEG2k liposomes (n ) 5)
64Cu-CB-TE2A-PEG2k liposomes (n ) 4)
%ID/g ( SD
A
%ID/g ( SD
C
%ID/g ( SD
B
blood
spleen
lungs
diaphragm
heart
8.71 ( 0.38
12.26 ( 0.70
3.14 ( 0.25
1.28 ( 0.16
2.89 ( 0.50
6.88 ( 0.97
6.14 ( 0.43
6.03 ( 0.52
4.83 ( 0.57
3.66 ( 0.63
0.31 ( 0.06
n.s.
*
n.s.
n.s.
n.s.
**
8.35 ( 1.11
20.25 ( 2.50
3.57 ( 0.20
1.41 ( 0.15
3.34 ( 0.74
15.15 ( 1.24
7.85 ( 1.17
7.60 ( 1.20
5.63 ( 0.14
4.55 ( 0.61
0.31 ( 0.09
n.s.
**
**
n.s.
*
**
*
*
6.85 ( 1.47
30.41 ( 1.40
2.24 ( 0.27
1.18 ( 0.43
2.21 ( 0.40
22.92 ( 1.94
5.80 ( 0.34
5.63 ( 0.33
4.74 ( 0.23
2.79 ( 0.99
0.27 ( 0.03
n.s.
***
*
n.s.
n.s.
***
n.s.
n.s.
n.s.
n.s.
n.s.
liver
left kidney
right kidney
duodenum
jejunum
muscle
*
n.s.
n.s.
n.s.
n.s.
n.s.
*
n.s.
a One-way analysis of variance followed by Tukey’s multiple comparison test was performed. (A: 64Cu-TETA liposomes vs 64Cu-TETA -PEG2k
liposomes; B:64Cu-TETA liposomes vs 64Cu-CB-TE2A-PEG2k liposomes; C:64Cu-TETA-PEG2k liposomes vs 64Cu-CB-TE2A-PEG2k liposomes, n.s.,
not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
Table 2. One-Phase Exponential Decay Curvesa of Liposomal
Clearance from the Blood Pool in Mice
64 transchelation with ceruloplasmin and superoxide dismu-
tase (SOD) with CB-TE2A as the Cu-64 chelator, we had
b
hypothesized that the accumulation of Cu-64 in the liver and
spleen would be lower than that observed using TETA (51, 52).
The TAC obtained here illustrated that liver accumulation
at 48 h (P < 0.001) was higher for 64Cu-CB-TE2A-PEG2k
liposomes than for 64Cu-TETA and 64Cu-TETA-PEG2k
liposomes (Figure 8c). The lipophilicity of 64Cu-labeled CB-
TE2A and negative charge of 64Cu-TETA may contribute to
the comparatively larger RES accumulation of 64Cu-CB-
TE2A-PEG2k liposomes. Due to the slow clearance and high
uptake of 64Cu-CB-TE2A-PEG2k liposomes in the liver
(Figure 8c) and spleen (Figure 8d), the decay-corrected whole
body activity (64Cu-CB-TE2A-PEG2k liposome) at 48 h was
also higher than other formulations (64Cu-CB-TE2A-PEG2k
liposomes vs 64Cu-TETA liposomes, p < 0.001; 64Cu-CB-
TE2A-PEG2k liposomes vs 64Cu-TETA-PEG2k liposomes,
p < 0.01; Figure 8a).
liposomes
t1/2 (h) span
k
64Cu-TETA liposomes (n ) 4)
18.22
18.15
16.20
41.10 0.03805
39.82 0.03819
38.32 0.04280
64Cu-TETA-PEG2k liposomes (n ) 5)
64Cu-CB-TE2A-PEG2k liposomes (n ) 4)
a Exponential decay curves were obtained from TAC of heart (Y )
span × e-k×t). b Half clearance time.
labeled liposomes yielded 40% of the initial blood-pool
activity at 24 h (27). In our study, the TAC data simulated
with a one-phase exponential decay showed that the 60%
clearance times were 24 h (64Cu-TETA liposomes), 24 h
(64Cu-TETA-PEG2k liposomes), and 21 h (64Cu-CB-TE2A-
PEG2k liposomes) (Figure 8b). There was no significant
difference in the time required for 60% clearance between
64Cu-TETA liposomes and 64Cu-TETA-PEG2k liposomes.
Given the slow release and the anticipated reduction in Cu-

Liposomal Cu-64 Labeling Using Bifunctional Chelators
Bioconjugate Chem., Vol. 21, No. 7, 2010 1213
Similar to the TAC data, the liver (P < 0.001) and spleen
(P < 0.001) biodistribution at 48 h (Table 1) showed that
the properties of all three liposomal formulations differed,
while a small but insignificant difference in accumulation
was observed in the kidneys, lung, heart, blood, intestine,
and muscle. At 48 h postinjection, liposomal accumulation
of 64Cu-TETA liposomes in the liver was 3.3-fold lower as
compared with that of 64Cu-CB-TE2A-PEG2k liposomes (P
< 0.001), and 2.2× less than that of 64Cu-TETA-PEG2k
liposomes (P < 0.001). Accumulation in the spleen also varied
in the order 64Cu-CB-TE2A-PEG2k liposomes > 64Cu-TETA-
PEG2k liposomes > 64Cu-TETA liposomes (Table 1). Ac-
cording to the previous literature, 64Cu-CB-TE2A showed
lower uptake in the liver and higher resistance to transche-
lation than 64Cu-TETA (51, 52), but these reports involve
the pharmacokinetics of chelators with (53) or without (52)
conjugation to a peptide. The comparison of 18F-labeled RGD
peptides with a PEG spacer ([18F]FB-PEG-RGD) or without
PEG ([18F]FB-RGD), reported by Chen et. al, did not show
a difference in accumulation of radioactivity in the liver,
although the blood clearance of [18F]FB-PEG-RGD was
slower than that of [18F]FB-RGD (4, 54). In contrast, the
liver and spleen accumulation of [125I]PEG-DPDPE was
greater than that of [125I]DPDPE (55). In ref 54, F-18 was
labeled at the end of the PEG chain and, in ref 55, I-125
was labeled on the tyrosine residue between DPDPE and the
PEG chain. Both results indicate that the presence of a PEG
spacer is not necessarily correlated with the accumulation
of radioactivity in the liver and spleen. The labeling position
and molecular structure is also likely to affect the RES
accumulation of activity. In our study, the radiolabel was at
the end of the lipid or PEG.
The differences in liver radioactivity observed here
between formulations may in part be due to the protective
effect of the PEG2k spacer brush while liposomes are passing
through the perisinusoidal space and sinusoids. In our
previous liposomal study using 6-BAT-lipid with PEG1.2k
as a spacer between the lipid and the chelator, 6.21 (
0.66%ID/g remained in the liver and 13.61 ( 6.19 in the
spleen at 48 h (36); here, with 64Cu-TETA liposomes, 6.88
( 0.97%ID/g remained in the liver and 12.26 ( 0.70%ID/g
in the spleen (Table 1). Those values are smaller than
radioactivity in the liver and spleen resulting from both 64Cu-
TETA-PEG2k and 64Cu-CB-TE2A-PEG2k liposomes (Table
1). 64Cu-chelator complexes of 6-BAT liposomes (36) and
64Cu-TETA liposomes are likely to be hidden under the
PEG2k brush, while 64Cu-chelator complexes of 64Cu-TETA-
PEG2k liposomes and 64Cu-CB-TE2A-PEG2k liposomes are
exposed at the end of the PEG2k brush.
Liposomes labeled with the BFCs were stable in the blood pool
over 48 h, and the 60% clearance time was 21-24 h. The
presence of the PEG spacer between the chelator and the lipid
did not significantly alter the labeling efficiency and the
clearance rate of liposomes from the blood pool. The location
of the 64Cu-chelator complex influences the liposomal uptake
in liver and spleen. The bifunctional chelator as a postlabeling
method is applicable for liposomal labeling as well as labeling
of antibodies and other nanoparticle formulations.
ACKNOWLEDGMENT
We thank the staff in the CMGI (Center for Molecular and
Genomic Imaging, UC Davis) for handling 64Cu and imaging
studies. We appreciate the support of NIH R01 CA016861, R01
CA103828, and R01 CA134659, and NCI 5R01 CA93375.
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