Biotinylated polyacrylamide-based metal-chelating polymers and their influence on antigen recognition following conjugation to a trastuzumab fab fragment

Article abstract of DOI:10.1021/bm300843u

We report the synthesis and characterization of metal-chelating polymers (MCPs) with a terminal biotin and a polyacrylamide backbone harboring multiple diethylenetriaminepentaacetic acid (DTPA) chelating sites. These polymers are conjugated to a streptavi

Full text of DOI:10.1021/bm300843u

Article
pubs.acs.org/Biomac
Biotinylated Polyacrylamide-Based Metal-Chelating Polymers and
Their Influence on Antigen Recognition Following Conjugation to a
Trastuzumab Fab Fragment
Peng Liu,† Amanda J. Boyle,‡ Yijie Lu,† Raymond M. Reilly,*,‡,§ and Mitchell A. Winnik*,†
†Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario, Canada M5S 3H6
‡Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2
§Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9
S
* Supporting Information
ABSTRACT: We report the synthesis and characterization of metal-chelating
polymers (MCPs) with a terminal biotin and a polyacrylamide backbone
harboring multiple diethylenetriaminepentaacetic acid (DTPA) chelating sites.
These polymers are conjugated to a streptavidin (SAv)-modified Fab fragment of
trastuzumab (tmFab) and subsequently complexed with 111In through DTPA.
Trastuzumab has specific targeting ability toward human epidermal growth factor
receptor-2 (HER2), which is overexpressed on some types of breast cancer cells
and ovarian cancer cells. 111In can generate Auger electrons which cause lethal
DNA double strand breaks. The radioimmunoconjugates (RICs) were designed to
target HER2 overexpressing cancer cells and carry multiple copies of 111In to these
cells. The mole maximum specific activities of these polymers were investigated by
loading the polymers with 111In at an increasing 111In to polymer ratio. The
polymers show 55-fold to 138-fold higher maximum specific activity than DTPA
modified tmFab-SAv. Moreover, the HER2 immunoreactivities of these RICs were
evaluated by measuring their specific binding ability toward HER2 overexpressing SKOV-3 ovarian cancer cells. The results
demonstrate that although in the presence of polymer there is increased nonspecific binding, HER2 targeting ability was retained,
ensuring the radionuclide delivery ability of these RICs.
MCPs. For example, a number of authors have introduced
pendant groups such as bipyridine7 or terpyridine8,9 as metal
binding ligands to promote supramolecular assembly of
polymers in solution10 or as coupling agents for noncovalent
curing applications.11 These new synthesis techniques allow the
preparation of block copolymers with the metal-chelating
groups confined to one of the blocks.12 The pendant-group
ligands can serve as antennas for light absorption and transfer
energy to chelated metal ions, leading to interesting studies of
the optical and electrochemical properties of these polymers.
For example, the Tew group has developed a series of polymers
with terpyridine as ligands to chelate lanthanide ions and
investigated their applications as optoelectronic materials and
as sensors for nerve gas detection.13,14
We have a twofold interest in developing MCPs for
biomedical applications. These polymers are designed to have
a narrow distribution of chain lengths, multiple sites for metal
chelation, and end-group functionality for attachment to
antibodies or other bioaffinity agents. First, we develop polymer
reagents for the new technique of mass cytometry. Mass
INTRODUCTION
■
Over the past twenty years there has been growing interest in
metal chelating polymers (MCPs). The early work by Torchilin
explored applications in biology and medicine, particularly the
development MCPs as carriers for Gd3+ as contrast enhance-
ment agents for magnetic resonance imaging.1 Most of these
experiments employed polylysine as a framework to which
mulitdentate chelators such as diethylenetriaminepentaacetic
acid (DTPA) were attached. His review in 1991 provides a
useful overview of early contributions to this field.2 In a 1999
paper,3 he showed that these DTPA-containing polylysines
could bind a larger number of 111In3+ ions than a biotin DTPA
derivative, and he suggested that this kind of polymeric reagent
would offer advantages over small molecule chelators in terms
of both γ-ray imaging and radiotherapy applications.
Over the intervening years, new techniques in synthesis
(controlled radical polymerization,4 ring-opening metathesis
polymerization,5 and better control over ring-opening polymer-
ization of amino-acid N-carboxyanhydrides6) have led to
polymers with a narrow distribution of chain lengths and
control over end functionality. In addition, advances in polymer
characterization techniques have given new confidence in one’s
ability to properly characterize polymers synthesized in this
way, and this in turn has led to interest in new applications of
Received: May 30, 2012
Revised: July 18, 2012
Published: August 7, 2012
© 2012 American Chemical Society
2831
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Figure 1. Concept of the experiment: (1) A tmFab-SAv conjugate is allowed to interact with and bind a biotin-end-capped metal-chelating polymer.
(2) The tmFab-SAv-MCP complex is incubated with 111In. (3) HER2+ cells are treated with the tmFab-SAv-MCP-111In complex in order to test
antigen recognition.
cytometry is a technique based on inductively coupled plasma
mass spectrometry (ICP-MS) for the rapid, highly multiplexed,
analysis of biomarker expression on individual cells.15 In this
technique, a suspension of cells is treated with a cocktail of
monoclonal antibodies (mAbs), each type labeled with a
different element or isotope. These cells are then nebulized
individually but stochastically into the argon plasma torch of an
ICP-MS with time-of-flight (TOF) detection. In the plasma,
cells are vaporized, atomized, and ionized to create an ion cloud
for which 20−30 TOF mass spectra are taken. The intensity of
signals for each isotope is a measure of the number of antigens
present in each cell. The role of the MCP is to increase the
number of metal ions of a given isotope bound to each type of
mAb.16 For example, we recently described an acrylamide-based
MCP with DTPA pendant groups and a maleimide end group.
The polymer could be attached to a mAb via Michael addition
of −SH groups generated by selective reduction of disulfide
bonds in the hinge region of the mAb. For this particular
example, an MCP with a number average degree of
polymerization (DPn) of about 80, we demonstrated that
each mAb contained on average 160 4 Tb ions with ca. 2.4
polymer molecules per mAb.17 We have used these polymer−
mAb conjugates to carry out an 11-plex assay of the cells
present in human cord blood.17 Nolan’s group at Stanford has
reported a 34-plex mass cytometric assay of signaling pathways
in the immune system.18
transfer due to their nanometer to micrometer range. Release of
Auger electrons in close proximity to the cell nucleus causes
lethal DNA double-strand breaks. 111In-NLS-trastuzumab is
bound, internalized, and transported to the nucleus of HER2+
BC cells, likely due to interaction of the NLS peptides with
importins α/β.20 111In-NLS-trastuzumab was 8-fold more
potent than trastuzumab at decreasing the clonogenic survival
of HER2-amplified SKBR-3 human BC cells,19 and 111In-NLS-
trastuzumab was able to kill trastuzumab-resistant BC cells,
provided that the HER2 extracellular domain (ECD) was
preserved.21,6 Nonetheless, the potency of 111In-NLS-trastuzu-
mab was highly dependent on HER2 density, exhibiting its
greatest effectiveness on SKBR-3 cells (1.3 × 106 HER2/cell).
It was significantly less effective on MDA-MB-361 cells with
intermediate HER2 density (5.1 × 105 HER2/cell), and MDA-
MB-231 cells with low HER2 expression (5.4 × 104 HER2/
cell) were resistant to 111In-NLS-trastuzumab.19
Our interest in developing MCPs for conjugation to
trastuzumab is based on the hypothesis that increasing the
amount of 111In delivered per HER2 recognition event would
enhance the potency of 111In-NLS-trastuzumab for killing
HER2-overexpressing BC cells and extend its range to BC cells
with lower HER2 density. This idea can be appreciated by the
fact that, at the specific activity (SA) achieved for 111In-NLS-
trastuzumab monosubstituted with DTPA (<0.24 MBq/μg;
<3.6 × 104 MBq/μmol), only 1 in 50 molecules was
radiolabeled.19 Thus, a high proportion of HER2 was bound
by nonradiolabeled immunoconjugates, limiting the cytotoxic
potency, particularly for cells with low HER2 expression.
Attaching a polymer with multiple chelators has the potential
to increase the number of radionuclides carried by each
antibody. Torchilin described a biotinylated polylysine modified
with up to 20 DTPA groups and suggested it as a means of
increasing the delivery of 111In to tumors pretargeted with
streptavidin (SAv)-conjugated antibodies.3 Here we describe
the synthesis of three polymers by reversible addition−
Here we are interested in developing MCPs as carriers for
radionuclides in targeted delivery to cancer cells, particularly
human epidermal growth factor receptor-2 positive (HER2+)
breast cancer (BC) cells. This work is an outgrowth of the
Reilly group’s studies of Auger electron radioimmunotherapy of
HER2+ BC based on the antibody trastuzumab (Herceptin)
modified with nuclear localization sequence (NLS) peptides
and derivatized with DTPA for labeling with 111In.19
Trastuzumab targets HER2 and Auger electrons are very low
energy (<25 keV) electrons that have high linear energy
2832
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
acid) PMAA standards. Samples were dissolved in deionized (DI)
water prior to injection.
fragmentation transfer (RAFT) polymerization that share a
common structure and carry a DTPA chelator on each pendant
group. The polymers differ in length and were designed to
examine how the length of the MCP affects antigen recognition
when attached to an antibody. To enable rapid scanning of a
broad family of different MCPs, the polymers carry a biotin end
group. These are then conjugated to a trastuzumab Fab
(tmFab) fragment covalently attached to a streptavidin (SAv)
via a polyethylene glycol (PEG) spacer.
Recently, a variety of polymers generated by RAFT
polymerization have been developed for biomedical applica-
tions.22 As in other living/controlled radical polymerization
methods, RAFT provides excellent control of polymer
molecular weight with narrow molecular weight distribution.
Moreover, its versatility for polymer end group functionaliza-
tion makes RAFT particularly promising in fields such as siRNA
delivery,23,24 anticancer drug delivery,25−27 as well as non-
invasive biomedical imaging.28
The basic philosophy of our experimental design for rapid
screening of different MCPs is presented in Figure 1. We
anticipate the synthesis of a broad family of different biotin-
end-capped MCPs (Bi-MCP). A tmFab-SAv conjugate can be
treated with a Bi-MCP, allowing one or more polymer
molecules to bind. The tmFab-SAv-Bi-MCP complex can be
labeled with 111In to create the radioimmunoconjugate (RIC):
tmFab-SAv-Bi-MCP-111In. This RIC would then be tested for
HER2 recognition on SKOV-3 (or other HER2 overexpress-
ing) cancer cells. One can construct and examine a large family
of Bi-MCPs to assess structure−chain−length−function
relationships in terms of ability to recognize HER2 on the
surface of BC cells. In this way, one can identify candidate
MCPs to develop for covalent conjugation to trastuzumab itself
for in vitro and in vivo studies.
In this paper our emphasis is on the synthesis and
characterization of the biotin-end-labeled MCPs, as well as on
the influence of polymer chain length on the antigen
recognition efficiency of the tmFab-SAv RIC. We describe
RAFT polymerization to generate polymer precursors with
biotin end functional groups, followed by postpolymerization to
quantitatively introduce DTPA pendant groups as binding sites
for 111In. These polymers were then bound to tmFab-SAv
through the strong interaction between biotin and SAv (Ka =
1015 M−1). The immunoreactivity of these RICs was then
evaluated through cell binding experiments. A detailed study of
the biodistribution in mice of tmFab-SAv RICs of a family of
biotin-end-labeled MCPs of similar length, including one of the
samples described here, is reported elsewhere.29
1H NMR spectroscopy was conducted with a Varian 400
spectrometer operating at 400 MHz. Mass analyses were obtained
using a QStar model quadrupole time-of-flight mass spectrometer (AB
Sciex, Concord, ON). Samples were run via loop injection using
positive electrospray ionization with the mobile phase composed of
1:1 (v/v) CH3OH/0.1% aqueous HCOOH. UV/vis measurements
were carried out with a Perkin-Elmer Lambda 6 spectrometer.
Thermal gravimetric analysis (TGA) measurements were carried out
with a TA Instruments SDT T600 instrument in the step-scan mode.
Carefully weighed polymer samples were heated at 150 °C for 1 h to
remove moisture and then at 600 °C to pyrolyze the sample,
converting all nonvolatile material to Na2CO3. From the ceramic yield,
the mean numbers of Na+ ions per DTPA group were calculated.
High pressure liquid chromatography (HPLC) analysis was
performed on a size-exclusion (SE)-HPLC on a BioSep SEC-S4000
column (300 mm × 4.6 mm; Phenomenex, Torrance, CA, USA). 100
mM NaH2PO4 buffer, pH 7.0, was used as eluent at a flow rate of 0.35
mL/min using a Series 200 pump (PerkinElmer, Wellesly, MA, USA)
interfaced with a diode array detector (PerkinElmer) set at 280 nm.
For each injection, 20 μL of 0.5 μg/μL solution radiolabeled with 0.4
MBq 111In was employed.
Materials. Except where noted, the following reagents were
purchased from Sigma Aldrich and used without further purification:
2,2′-(ethylenedioxy)diethylamine (MW = 148, 98%), N,N′-dicyclo-
hexylcarbodiimide (DCC, 99%,), dimethylformamide (DMF, 99%),
ethyl acetate (HPLC grade, from Fisher Scientific), trifluoroacetic
anhydride (TFAA, >99%), N-(tert-butoxycarbonyl)-1,2-diaminoethane
(tBoc-DE, from TCI, >97%), trifluoroacetic acid (TFA), tetrahy-
drofuran (THF, >99%), triethylamine (TEA, >99%), DTPA > 98%,
azobis(4-cyanovaleric acid) (ACVA, >98%, from Fluka), pentafluor-
ophenol (pfp-OH, >99%, from Matrix Scientific Inc.), anhydrous
pyridine (>99%,), biotin (>99% from Nanjing Tianzun Zezhong Inc.,
China), 1,2-lutidine (>96%, from Fluka), 2,2′-azobis(2-methylbutyr-
onitrile) (AMBN, from Dupont USA), and 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, from Acros
Organic, >99%, Fisher Scientific, Canada). TEA was distilled from
KOH, dichloromethane (DCM, Aldrich) was distilled from CaH2, and
dithiobenzoic acid disulfide was prepared as described previously.30
Other items include silica for chromatography (Merck grade 9385,
230−400 mesh, 60 Å), a Spectra/Por dialysis membrane (MWCO 1
kDA, Spectrum Laboratories, Inc.), and a Millipore Amicon spin filter
(3 kDa MWCO, 4 mL, Fisher Science, Canada). The tmFab and
tmFab-SAv were prepared and characterized as described previously.29
111InCl3 was purchased from Nordion, Inc. (Kanata, Canada).
Synthesis of the Acrylamide Monomer N-{2-[(BOC)-
aminoethyl]} Acrylamide (1). tBOC-DE (4.00 g, 25.0 mmol),
TEA (3.80 mL, 27.3 mmol), and THF (20 mL) were placed into a 500
mL round-bottom flask (RBF). Acryloyl chloride (2 mL, 25 mmol)
was dissolved in THF (180 mL) and added dropwise into the RBF
with stirring and cooled with an ice-bath, over 2 h. The solution was
allowed to react for another 2 h, during which time some white solids
precipitated (salts of TEA), which were removed by filtration. Then
the THF was removed with a rotary evaporator. The raw product was
dissolved in chloroform (100 mL) and extracted with brine (2 × 100
mL). The organic phase was collected and dried over magnesium
sulfate overnight; then after filtration, the solvent was removed with a
rotary evaporator. The raw product was purified by chromatography
over silica (300 mL) using chloroform/methanol = 20:1 (Rf ≈ 0.3, 1.6
L) as the eluent. The product was obtained as a white solid (3.80 g,
71.8%). 1H NMR (400 MHz, CDCl3, δ (ppm), cf. Figure S1,
Supporting Information): 6.25 (t, 1 H, −CH=), 6.09 (t, 1 H, =CHH),
5.51 (t, 1 H, =CHH), 4.91, 6.38 (s, 2 H, −NH), 3.25, 3.41 (t, 4 H, N−
CH2), 1.28 (s, 9 H, C(CH3)3).
EXPERIMENTAL SECTION
■
Instrumentation. Size exclusion chromatography (SEC) measure-
ments on samples dissolved in N-methylpyrrolidone (NMP) were run
on a system consisting of a Viscotek VE1121 solvent pump, an
American Standard Corporation gel 10 μm column, and a gel 10 μm
guard column and a Viscotek VE 3580 refractive index detector. The
analysis was carried out at 80 °C at a flow rate of 0.6 mL/min with
NMP containing 0.2 wt % LiCl as the eluent, and the column was
calibrated with poly(methyl methacrylate) (PMMA) standards. Water-
soluble polymers were analyzed with a Viscotek SEC consisting of a
Viscotek VE1122 solvent delivery system and a VE7510 GPC
degasser, with Viscotek ViscoGEL G4000PWXL and G2500PWXL
columns (kept at 30 °C) connected to a Viscotek VE3210 UV/vis
detector and a VE3580 refractive index detector. The eluent was 0.2 M
KNO3, 200 ppm NaN3, and 25 mM, pH 8.5 phosphate buffer at a flow
rate of 1.0 mL/min. The system was calibrated with poly(methacrylic
Synthesis of the Biotin Chain Transfer Agent (CTA) 2.
Synthesis of Biotinylpentafluorophenyl Ester (Bi-PFP). Bi-PFP was
synthesized as described by Korshun et al.31 Biotin (2.00 g, 8.2 mmol)
was placed into a 250 mL RBF, and anhydrous pyridine 50 mL was
added. The solution was refluxed for 2 h until all the biotin dissolved.
2833
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Table 1. Ratio of Reagents Used in the RAFT Polymerization of 1 and Characteristics of the Polymers Obtained
a
b
c
d
e
[C]/[M]
[C]/[I]
reaction time (h)
DPn
Mn(NMR)
Mn(SEC)(PDI)
Bi-PBocNAAm-20
Bi-PBocNAAm-40
Bi-PBocNAAm-50
1/40.0
1/79.6
1/80.0
3.98/1
4.00/1
4.00/1
4
6
8
20.3 1.4
39.7 2.8
50.8 3.6
5.0 kDa
9.1 kDa
4.3 kDa (1.16)
11.9 kDa (1.21)
12.8 kDa (1.36)
f
11.5 kDa
a
b
[C]/[M] molar ratios of chain transfer agent to monomer used in the synthesis. [M]/[I] molar ratios of chain transfer agent to initiator used in
c
1
the synthesis. Number average DPn as determined by H NMR. The standard deviation in DPn was estimated assuming a 5% error in the
d
integration of the aromatic and aliphatic 1H signals being compared. Mn(NMR) values were calculated by multiplying DPn by the molecular weight
e
of the monomers plus the molecular weight of the chain transfer agent end group. Mn(SEC) refers to nominal Mn values determined by SEC in
f
NMP. These values and values of PDI = Mw/Mn were determined by reference to PMMA standards. Using the CTA (2) (ε300 = 16.8 M−1 cm−1) as
a reference, we determined a value of Mn(UV) = 8.2 kDa.
1
Figure 2. H NMR spectrum of biotin-PBocNAA-40 in methanol-d4 with one drop of D2O added.
After the solution was cooled to room temperature (RT), PFP-OH
(1.54 g, 8.4 mmol) was added into the flask. The temperature was
reduced to 0 °C. Then DCC (1.80 g, 8.7 mmol) dissolved in
anhydrous pyridine (12 mL) was added dropwise into the flask with
stirring. The flask was incubated in an ice-bath for 2 h. Then the ice-
bath was removed and the solution was stirred for another 16 h. The
solution was then filtered to remove some of the byproduct,
dicyclohexyl urea. Half the solvent was then evaporated on a rotary
evaporator, and the solution was allowed to stand for 5 h at RT. The
solution was filtered to remove the precipitate, and then most of the
solvent was removed by rotary evaporation. The syrup was added
dropwise into a mixture of hexane/chloroform (1:1), (200 mL). The
precipitate was redissolved in pyridine and washed again with the
mixture of hexane and chloroform. The solid was filtered and dried in
was precipitated into diethyl ether. Then the solid was dissolved in
DMF (20 mL) and precipitated again in diethyl ether (150 mL). This
product was purified by flash chromatography (400 mL silica)
(ethanol/ammonia hydroxide (28%) = 5:1). The solvent was removed
1
in vacuum, and a white solid was obtained (1.14 g, 83%). H NMR
(400 MHz, deuterium oxide, δ (ppm) cf., Figure S2): 4.48, 4.30 (m, 2
H, CHNH), 3.56 (s, 4 H, OCH2CH2O), 3.48 (m, 4 H, OCH2), 3.26
(m, 2 H, CH2NH), 3.21 (m, 1 H, CHS), 2.87 (dd, 1 H, CHHexoS),
2.72 (t, 2 H, CH2-NH-CO-biotin), 2.65 (d, 1 H, CHHendoS), 2.15 (t, 2
H, biotin-CH2CO), 1.2−1.6 (m, 6 H, biotin-CH2). MS (ESI m/z, [M
+ H]+ = 375.2).
Synthesis of Bis(pentafluorophenyl)azobis(4-cyanovalerate)
(Bis-PFP-ACV) and Pentafluorophenyl[4-(phenylthiocarbo-
nylthio)-4-cyanovalerate] (CTA-PFP). These syntheses followed
the procedures reported by Roth et al.33 ACVA (5.14 g, 18.3 mmol),
pfp-OH (7.90 g, 42.9 mmol), 2,6-lutidine (14.91 g, 139.2 mmol), and
dry DCM (105 mL) were placed in a 250 mL RBF. The flask was
cooled to 0 °C with an ice bath, and the mixture was bubbled with
nitrogen for 10 min. Then TFAA (11.30 g, 53.8 mmol) was injected
through a septum. The solution turned yellow. The ice bath was
removed. As the reaction was stirred for an additional 24 h, the
solution turned brown. Then the solution was extracted with water (2
× 100 mL) to remove the TFAA. The solution was concentrated with
a rotary evaporator and precipitated into hexane (70 mL). A white
1
vacuum. A white solid was obtained (2.60 g, 78%). H NMR (400
MHz, d6-DMSO, δ (ppm)): 6.48, 6.41 (2 H, NH); 4.34, 4.16 (m, 2 H,
CHNH); 3.16−3.11 (m, 1 H, CHS); 2.85−2.81 (dd, 1 H,CHHexoS);
2.77 (t, 2 H, CH2CO); 2.60 (d, 1 H, CHendoHS); 1.69−1.41 (m, 6
+
H, CH2-biotin). MS (ESI m/z, [M + H]+ = 411.1, [M + Na]
=
433.1)
Synthesis of N-1[-2,2′-(Ethylenedioxy)diethyl-1′-amino]-
biotinylamide (Bi-EO2NH2). 2,2′-(Ethylenedioxy)diethylamine (6.2
mL, 37.0 mmol) and anhydrous TEA (1.5 mL, 11 mmol) were
placed in a 250 mL RBF.32 Bi-PFP (1.50 g, 3.7 mmol) was dissolved in
anhydrous DMF (75 mL) and added dropwise into the flask over 1 h.
The solution was stirred magnetically for another hour. Most of the
solvent was removed with a rotary evaporator, and the raw product
1
solid was obtained: yield 9.94 g (88%) after drying in vacuum. H
NMR (400 MHz, CDCl3, δ (ppm)): 3.00−2.48 (m, 8 H, CH2), 1.78,
1.73 (2 s (cis, trans), 6 H, CH3).
2834
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Table 2. Mn Values, Water, and Na+ Ion Content Determined by TGA Analysis, and Effective Molecular Weights of the Three
Bi-PAAm(DTPA) Polymers
a
b
c
d
e
Na+ ions per DTPA
effective Mn (kDa)
NMR
NMR
SEC
DPn
Mn
(kDa)
Mn (kDa) (PDI)
H2O per DTPA
Bi-PAAm(DTPA)-20
Bi-PAAm(DTPA)-40
Bi-PAAm(DTPA)-50
20.4 1.4
10.0 0.7
19 1.0
23 1.2
14.8 (1.2)
27.0 (1.2)
42.7 (1.4)
3.2 0.16
2.1 0.11
2.6 0.13
2.8 0.14
3.1 0.16
12.8 1.0
25.3 2.0
31.7 2.5
f
40 2.0
f
51 2.6
3.6 0.18
a
b
Calculated from DPn, assuming that the DTPA groups are in the fully protonated form. Nominal Mn values and PDI (Mw/Mn) are from aqueous
c
SEC measurements using poly(methacrylic acid) (PMAA) standards. Determined from the mass loss at 100 °C in TGA measurements.
d
e
1
Determined from the ceramic yield (Na2CO3) at 600 °C in TGA measurements. Calculated from the number average DPn determined by H
f
NMR and includes the mean number of Na+ ions and water molecules per repeat unit. These values are taken from Table 1 and refer to values
determined for the RAFT polymers 3.
Bis-PFP-ACV (3.10 g, 5.1 mmol), dithiobenzoic acid disulfide (1.06
g, 3.4 mmol), and ethyl acetate (50 mL) were placed into a 250 mL
RBF, which was then capped with a septum and bubbled with nitrogen
for 20 min. The reaction mixture was refluxed for 16 h. Then the
solvent was removed on a rotary evaporator, and the remaining solid
was purified by flash chromatography (hexane/chloroform = 4:1, silica
400 mL). Using 3 L eluent, we obtained 2.83 g of CTA-PFP (63%).
1H NMR (400 MHz, CDCl3, δ (ppm), cf., Figure S3A): 7.91 (d, 2 H,
o-Ar), 7.57 (t, 1 H, p-Ar), 7.39 (t, 2 H, m-Ar), 3.08−3.01 (m, 2 H, O
C−CH2), 2.80−2.70 (m, 1 H, OCH2−CHH), 2.59−2.49 (m, 1 H,
OCH2−CHH), 1.97 (s, 3 H, CH3). MS (ESI m/z, [M + H]+ =
446.0).
[monomer 1 (1.50 g, 7.0 mmol), chain transfer agent 2 (112.4 mg,
0.18 mmol), and AMBN (8.45 mg, 0.044 mmol)]. The shortest chain
polymer was purified by transferring a solution in methanol into a 1
kDa dialysis membrane and dialyzing against methanol for 5 days. The
1H NMR spectra for these two polymers are presented in Figures S4A
and S4B.
Pendant Group Transformations. Removal of the RAFT Agent
and Deprotection of the tBoc Groups. Polymer 3 was treated with
ethanolamine in THF to remove the RAFT agent, and then the free
−SH groups at the chain end were subjected to a Michael reaction
with excess 1. These polymers were immediately treated with a
mixture of DCM and TFA to remove the tBOC protecting groups.
Details of one example are provided below.
Synthesis of N-Biotinyl-N′-(3-(4-phenylthiocarbonylthio-4-
cyanovaleryl)-3,6-dioxaoctane-1,8-diamine CTA (2). This syn-
thesis followed a procedure similar to that reported by Bathfield et al.34
CTA-PFP (0.38 g, 0.85 mmol) was placed in a RBF and dissolved with
dry DCM (40 mL). Bi-PFP (0.30 g, 0.8 mmol) was suspended in dry
DCM (27 mL) and added to the flask in three portions at 20 min
intervals. After 4 h, the solution was extracted with water (3 × 70 mL).
All solvent was removed on a rotary evaporator. Then the raw
compound was purified by two consecutive column chromatography
steps (initially (silica 200 mL, DCM eluent 1 L), then DCM/methanol
= 20:1 (silica 200 mL, eluent 3 L)). The product (0.25 g, 50%) was
Bi-PBocNAAm (DPn = 40) (50.10 mg, 0.0057 mmol) was placed in
a 20 mL glass vial. The vial was capped with a rubber septum and
purged with nitrogen for 5 min. Then anhydrous THF (0.5 mL),
which had been purged with nitrogen for 5 min, was injected into the
vial to dissolve the polymer. After all the polymer had dissolved,
ethanolamine (3.5 μL, ca. 10 equiv to moles of polymer chains) was
injected into the vials for aminolysis of the dithiobenzoate groups.
After 30 min, the yellow color of the dithiobenzoate ester group
disappeared. Monomer 1 (18.01 mg, 0.084 mmol, ca. 15 equiv to
moles of polymer chains) was dissolved in anhydrous THF (0.2 mL),
which had been degassed with nitrogen, and this solution was injected
into the vial to react with −SH groups on the polymer chain ends. The
reaction was stirred for 4 h. After the solvent was removed with a
rotary evaporator, the solid was dissolved in methanol and dialyzed
against methanol in a 1 kDa MWCO dialysis bag for 2 days. Then the
solution was concentrated with a rotary evaporator. The solid was
dissolved in a mixture of DCM and TFA (7:3) (2 mL) and stirred for
4 h at RT to remove the tBOC groups. After that, the polymer was
precipitated into 20 mL of diethyl ether. The supernatant was
decanted, and the solid was further dried under vacuum. The amino
polymer was obtained as a white solid, as the polytrifluoroacetate salt
(38.14 mg, 67%).
Synthesis of Poly(acrylamide-diethylene-diamide-DTPA) [Bi-
PAAm(DTPA)]. DTPA (3.76 g, 9.55 mmol, about 80 equiv to the
amine groups on the polymer) was dissolved in 5 M aqueous NaOH,
adding enough base to adjust the pH to 8.5 (monitored with a pH
meter). DMTMM (251 mg, 0.91 mmol, ca. 8 equiv to the amine
groups on the polymer) was dissolved in DI water (2 mL) and added
into the DTPA solution. The solution was given 5 min to prereact.
Then a sample of the amino polymers described above (25.02 mg) was
dissolved in DI water (2 mL) and added into the solution containing
DTPA and DMTMM. The solution was stirred for 1 h, transferred to a
15 mL, 3 kDA MWCO Millipore Amicon spin filter, and washed with
water (9 × 11 mL). Finally, the aqueous solution was freeze-dried to
yield the biotin-PAAm (DTPA) polymer (40 mg).
1
obtained as a purple solid. H NMR (400 MHz, CDCl3, δ (ppm) cf.,
Figure S3B): 7.91 (d, 2 H, o-Ar), 7.57 (t, 1 H, p-Ar), 7.39 (t, 2 H, m-
Ar), 6.99 (t, 1 H, NHC(O)C2H4C(CN)(CH3)S), 6.20, 5.70 (s, 2 H,
NH), 4.82 (t, 1 H, NHC(O)(C4H8)), 4.48, 4.30 (m, 2 H, CHNH),
3.63−3.39 (m, 12 H, CH2CH2OCH2CH2OCH2CH2), 3.14 (m, 1 H,
CHS), 2.90 (dd, 1 H, CHHexoS), 2.76−2.54 (m, 4 H, OCH2−
CH2C(CN)(CH3)S), 2.43 (d, 1 H, CHHendoS), 2.22 (t, 2 H, biotin-
CH2CO), 1.95 (s, 3 H, CH3), 1.68−1.46 (m, 6 H, biotin-CH2). Elem.
Anal.: theor C 54.78, H 6.50, N 11.01; found C 53.63, H 6.58, N
10.60. MS (ESI m/z, [M + H]+) Cal: 636.2342. Exp: 636.2346.
Synthesis of Polymers through RAFT Polymerization. RAFT
Polymerization To Form Biotin-poly-(N-{2-[(BOC)aminoethyl]}
Acrylamide) (Bi-PBocNAAm). In these polymerizations, 1 was used
as the monomer and 2 was used as the chain transfer agent, with
AMBN as the initiator. Polymers with different chain lengths were
synthesized by varying the ratio of monomer to chain transfer agent
and by varying the reaction time (Table 1). A representative example
is described below.
Bi-PBocNAAm with DPn = 40. Monomer 1 (1.50 g, 7.0 mmol),
chain transfer agent 2 (55.62 mg, 0.088 mmol), and AMBN (4.19 mg,
0.022 mmol) were dissolved in dioxane (10 mL) and transferred to a
50 mL Schlenk flask. The solution was degassed by five freeze−
pump−thaw cycles and then sealed. The solution was stirred at 70 °C
for 4 h and then quenched by cooling the reaction flask with liquid
nitrogen. The polymer was purified by precipitating into a mixture of
hexane/diethyl ether (1:2, 40 mL). The solid obtained was redissolved
in THF and precipitated into 40 mL of a mixture of hexane and diethyl
Molecular Weight Determination. The number average DPn
values were determined by end group analysis using 1H NMR. For the
as-prepared RAFT polymer, we compared signals due to the CH2
groups of the biotin as well as the phenyl protons of the RAFT agent
to the tert-butyl groups of the repeat units. Polymers (10 mg) were
1
ether. This process was repeated five times. A H NMR spectrum of
this polymer is presented in Figure 2 of the Results and Discussion
section.
In a similar manner, polymers were synthesized with DPn = 50
[monomer 1 (2.00 g, 9.35 mmol), chain transfer agent 2 (74.28 mg,
0.11 mmol), and AMBN (5.58 mg, 0.029 mmol)] and with DPn = 20
1
dissolved into CD3OD (1 mL), and 2 drops of D2O were added. H
NMR spectra were acquired with 10 s delay times and 512 scans.
Curiously, spectra of polymers taken in a mixture of methanol-d4 and
2835
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Scheme 1. Synthesis of the Biotin Chain Transfer Agent 2
D2O show what appear to be two discrete -OH peaks at 4.6 and 4.8
ppm (Results and Discussion; Figure 2). Examination of the solvent
mixture itself also showed these two peaks in addition to the CHD2O-
signal at 3.3 ppm. Following subsequent transformations of the
polymer, values of DPn were estimated by comparing integrations of
the CH group of the biotin at the peak at 4.3 ppm to that of signals
characteristic of the pendant groups.
based on the effective molecular weight (Mn) of the MCPs. For HER2
binding experiments, 0.04 nmol of tmFab-SAv (4.4 μg) was incubated
with 0.04 nmol of Bi-PAAm(DTPA)-20, Bi-PAAm(DTPA)-40, or Bi-
PAAm(DTPA)-50 (0.5 μg, 1.0 μg, and 1.3 μg, respectively) in a final
volume of 15 μL of PBS for 30 min at RT.29 The resulting complexes
were buffer-exchanged into 100 mM sodium acetate buffer, pH 6.0, by
ultrafiltration on an Amicon Ultra device (MWCO = 50 kDa).
Radiolabeling with 111In and Determination of Maximum SA
Achievable. The maximum SA that could be achieved for labeling
with 111In was identified by incubating decreasing amounts of Bi-
MCPs (2, 0.2, 0.02, or 0.002 μg) or tmFab(DTPA)-SAv (10, 1.0, 0.1,
and 0.01 μg) incorporating 2.1 0.1 DTPA groups/molecule, with a
constant amount of 111InCl3 (3.7 MBq) in 100 mM sodium acetate,
pH 6.0, for 1 h at RT. The final volume was 15 μL. The percent
labeling efficiency (LE) was measured by instant thin layer silica gel
chromatography (ITLC-SG) in 100 mM sodium citrate, pH 5.0, as
previously reported.29 The Rf values of 111In-labeled Bi-MCPs or 111In-
tmFab(DTPA)-SAv and free 111In in this system are 0.0 and 1.0,
respectively. The SA obtained (MBq/μg and MBq/μmol) with the
minimum mass of Bi-MCP or tmFab(DTPA)-SAv that provided a LE
>80% was taken as the maximum SA practically achievable.
Determination of the maximum SA achievable for Bi-MCPs
complexed to tmFab-SAv was not performed, since this varies with
the number of Bi-MCPs complexed to the immunoconjugates due to
the tetravalency of SAv, and this was not known. For evaluation of
HER2 binding, tmFab-SAv-Bi-MCP complexes and tmFab-SAv
derivatized with DTPA were radiolabeled by incubation of 4 nmol
of each in 30 μL of 100 mM sodium acetate buffer, pH 6.0, with 2
MBq 111InCl3 for 1 h at RT, as previously reported.29 The samples
were then buffer-exchanged into PBS, pH 7.4, by ultrafiltration on an
Amicon Ultra device (MWCO = 30 kDa). The final radiochemical
purity was >95%, measured by ITLC-SG in 100 mM sodium citrate,
pH 5.0. The Rf values of the 111In-complexes and free 111In were 0.0
and 1.0, respectively.
Polymers were isolated by freeze-drying from solution at pH 8.5,
containing sodium ions as well as small traces of water. To calculate
micromoles of polymer for weighed samples, we needed to determine
an “effective molecular weight” for each sample. This was
accomplished by subjecting the polymers to thermal gravimetric
analysis (TGA) as described by Majonis et al.35 Carefully weighed
samples (ca. 5 mg) were heated at 100 °C to remove moisture and
then at 600 °C to convert the remaining polymer to Na2CO3. From
the ceramic yield, we determined that each polymer contained
between 2 and 3 Na+ ions per DTPA group. These values and other
characteristics of the polymers are collected in Table 2 below.
As in ref 17, standard error propagation expressions were used to
calculate the standard errors. We assumed an uncertainty of 5% in
NMR integrations and an inherent 5% error for each water/DTPA
value and sodium/DTPA value from the TGA measurements.
A value of Mn (UV) for Bi-PBocNAAm-40 was estimated as follows:
A sample of Bi-PBocNAAm-40 (2.14 mg) was dissolved in CHCl3 and
diluted to 25.0 mL. Two dilutions were also prepared; then UV spectra
were obtained using 1.00 cm cells. From the plot (not shown) of
absorbance at λmax = 300 nm vs polymer concentration, using ε300
=
1.68 × 104 M−1 cm−1 determined from CTA 2 as the model
compound, we calculated Mn (UV) = 8.2 kDa.
Preparation of Immunoconjugates. Conjugation of Trastuzu-
mab Fab Fragments to SAv. The details of covalent attachment of
SAv to trastuzumab Fab fragments (tmFab-SAv) and trastuzumab Fab
fragments with DTPA chelators (tmFab(DTPA)-SAv) are described
elsewhere.29 Briefly, SAv was pretreated with Traut's reagent to
convert a lysine amino group to a thiol for reaction with the maleimide
of a heterotelechelic oligoethylene glycol with a maleimide at one end
and an N-hydroxysuccinimide ester (NHS) at the other.29 This, in
turn, was reacted with a lysine amine on the Fab fragments. For
tmFab(DTPA)-SAv, the Fab fragment was pretreated with DTPA
dianhydride to introduce the chelator; then the streptavidin was
attached via an oligoethylene glycol spacer as described above.
Complexation of Bi-MCPs to tmFab-SAv. Experiments were
designed to obtain a molar ratio of tmFab-SAv/Bi-MCP of 1:1
HER2 Binding. HER2 binding of 111In-labeled tmFab(DTPA)-SAv
or tmFab-SAv complexed to Bi-MCPs was measured using SKOV-3
human ovarian cancer cells (1 × 106 HER2/cell). Briefly, 1.5 × 105
cells were cultured overnight in wells in a 24-well plate (Sarstedt, QC)
in RPMI-1640 growth media (Sigma-Aldrich, USA) containing 10%
fetal bovine serum. Following removal of the media, the cells in each
well were incubated with 0.5 mL of 10 nM of RICs (0.25 MBq/μg) in
serum-free medium for 3 h at 4 °C. Nonspecific binding was evaluated
by repeating the assay in the presence of a 100-fold excess of unlabeled
2836
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Scheme 2. RAFT Polymerization of Monomer 1 and Subsequent Transformation of the Polymer To Remove the
Dithiobenzoate End Group and To Introduce DTPA Chelating Groups on Each Repeat Unit
trastuzumab IgG. The media was removed, and the adherent cells were
rinsed with PBS, pH 7.4, and then solubilized in 100 mM NaOH at 37
°C for 30 min. The dissolved cells were transferred to γ-counting
tubes, and the total cell-bound radioactivity was measured in a γ-
counter. Specific binding was calculated by subtracting nonspecific
binding from total binding. HER2 binding was expressed as percentage
of RICs bound per 1.5 × 105 SKOV-3 cells.
necessary to assume that the modified polymers have DPn
values very similar to those of their precursors.
Synthesis of the Biotin Chain Transfer Agent. Our
synthesis of the biotin chain transfer agent is outlined in
Scheme 1. It is based on the idea of Bathfield et al.34 for
preparing a dithiobenzoate derivative that also contains an
active ester and reacting it with a biotin-amine reagent. We
employed CTA-PFP, first reported by Theato and co-
workers,33 and reacted it with Bi-EO2NH2. In this reaction
sequence, both the carboxylic groups of the biotin and ACVA
were activated by the formation of PFP activated ester groups.
While the Bi-PFP activated ester was synthesized effectively
through traditional DCC coupling chemistry (yield 78%), we
employed TFAA as a coupling agent, as performed previously
by Green et al.,36 to synthesize the PFP-activated ACVA (Bis-
PFP-ACV). Here the yield was somewhat higher (88%), and we
avoided the cumbersome removal of dicyclohexylurea as a
byproduct in the DCC coupling reaction. CTA-PFP itself was
synthesized by refluxing Bis-PFP-ACV with dithiobenzoate
disulfide in ethyl acetate. In the reaction of CTA-PFP with Bi-
EO2NH2, we chose to employ a small excess of CTA-PFP.
While the PFP ester is more reactive to amines than the
dithiobenzoate, we were concerned that an excess of amine
might lead to some aminolysis of the CTA. The biotin
functionalized CTA was then purified by column chromatog-
RESULTS AND DISCUSSION
■
Our philosophy for the synthesis of end-functional MCPs is
based on the idea that one should begin with the polymer-
ization of a readily available monomer to obtain a polymer for
which the number average of DPn can be determined with
confidence by 1H NMR. Once the base polymer is synthesized
and characterized, we carry out a series of pendant group
transformations, using reactions that occur in close to
quantitative yield, to introduce metal chelating groups such as
DTPA on each of the repeat units. Polydispersity can be
checked by SEC, and we monitor DPn by end group analysis
after each step. In our experience, however, pendant group
1
transformations introduce new signals to the H NMR. The
NMR spectra become more complex, and some of the new
signals may overlap signals associated with the end group. Thus,
it becomes more difficult to quantitatively compare the
integrals of peaks associated with the end group to those
associated with backbone or pendant group protons. This
problem is particularly acute here, where, in the initial polymer
synthesis, the dithiobenzoate end group allows values of DPn to
1
raphy. H NMR spectra of the biotin-CTA 2 and several of its
precursors are presented in Figures S2−S4.
RAFT Polymerization. The synthesis of the Bi-MCPs
described here began with RAFT polymerization of the
monomer N-{2-[(BOC)aminoethyl]} acrylamide (1) in
dioxane in the presence of the biotin-RAFT agent N-biotinyl-
N′-(3-(4-phenylthiocarbonylthio-4-cyanovaleryl)-3,6-dioxaoc-
tane)-1,8-diamine (2), initiated by AMBN. Our target was the
1
be determined with confidence by H NMR. But in later steps
of the synthesis, after removal of the dithiobenzoate, we have
only individual protons of a biotin end group as a reference
peak for estimating DPn by 1H NMR. Then it becomes
2837
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
synthesis of three samples of Bi-PBocNAAm with DPn of
approximately 20, 40, and 50.
The polymers were then treated with TFA in DCM to
remove the tBOC groups and to yield the deprotected polymer
5 as its polytrifluoroacetate salt. In the H NMR spectra, the
1
The reaction was stopped by cooling the reaction vessel and
then pouring the contents into a mixture of diethyl ether and
hexane to precipitate the polymer. Ether itself was ineffective
because the polymers show some solubility in this solvent. In
Table 1 we present the ratios of reactants used in these
syntheses and the values of the number average of DPn
peaks for the tert-butyl groups at ca. 1.4 ppm disappeared, and
two sharp peaks appeared at 2.9 and 3.3 ppm, representing the
CH2 groups on the pendant protonated aminoethyl amide
1
substituents. The H NMR spectrum of 5 (DPn = 40) is
presented in Figure S8. The pendant amino groups on these
polymers were converted to DTPA monoamide derivatives
following the procedure developed by Majonis et al.17 In this
approach, a large excess of DTPA is prereacted with a limiting
amount of the coupling agent DMTMM in aqueous solution at
pH 8.5. The polymer in water is then added, and the reaction is
complete in about an hour. These conditions are effective at
quantitative conversion of polymer primary amines to DTPA
monoamides without any detectable cross-linking that would
appear as peak broadening in SEC measurements. The
polymers were subsequently purified from water-soluble low
molecular weight side products and excess reactants by dialysis
or spin filters.39 The polymers were then isolated as powders by
lyophilization.
1
determined by H NMR. By varying the ratio of reactants
and reaction time, polymers with different chain lengths were
achieved, and the number average DPn values determined by
1H NMR corresponded closely with the target values. The
polymer with the DPn of 20 had to be purified by dialysis in
methanol because we were unable to find a ratio of hexane to
diethyl ether suitable to separate this short chain polymer from
unreacted monomer.
The 1H NMR spectrum of Bi-PBocNAAm-40 with DPn = 40
is presented in Figure 2. The signals from the protons on the
dithiobenzoate end groups appear at 7.91, 7.57, and 7.39 ppm.
To determine DPn, the integral of these signals was compared
with the integral of peaks appearing in the range 2.59−0.38
ppm. These peaks contain the signals of protons of the polymer
backbone (d and e in Figure 2) and protons from the tert-butyl
(Boc) groups (h). The signals in the region of ca. 3−4 ppm
represent protons on the ethylene diamine linkers overlapped
with the peaks of methanol protons. Peaks m, n, and i−l from
the biotin-EO2NH-end group are resolved in the spectrum, and
they confirm the presence of biotin groups on the polymers.
Analysis of all three RAFT polymers in this way gave the
values of DPn collected in Table 1. SEC traces for all three
polymers are presented in Figure S5 (Supporting Information).
Calculated values of Mn and the polydispersity index PDI based
on PMMA standards are also presented in Table 1.
SEC traces (in water) of the three polymers 6 obtained in
this way are presented in Figure 3, and values of Mn and PDI
Postpolymerization Modification. In Scheme 2, we
present the chemical reactions for the RAFT polymerization
and for all the postpolymerization modifications of the
polymers we prepared.
For the applications described below, we wanted to avoid any
complications that might be caused either by the chain transfer
group or by free thiol groups that might be liberated in situ due
to reactions of this group. Following the lead of others,37 we
removed the dithiobenzoate group of the RAFT polymer 3 by
aminolysis with ethanolamine and then trapped the free thiols
by Michael addition with the acrylamide monomer 1. In the 1H
NMR spectra of 4, we note the disappearance of the aromatic
protons of the chain transfer agent (Figure S6). We also
observed a new peak at δ = 2.9 ppm due to protons on the
carbon adjacent to the thiol ether end group.38 An example of
the polymer with DPn = 20 is shown in Figure S6. With the
removal of the dithiobenzoate group, it becomes significantly
more difficult to use 1H NMR to obtain reliable values of DPn.
Signals associated with the biotin end group can be resolved,
but it is difficult to compare the peak integrations of the
pendant group signals with those of the end group. At this stage
of the synthesis, we prefer to rely on SEC chromatograms to
monitor chain length and polydispersity. In SEC chromato-
grams (Figure S7), the transformation of 3 to 4 largely
preserved the shape of the peak for each polymer, with a small
peak shift for the sample with DPn = 20. These SEC traces
provide evidence that end group transformation did not
significantly change the chain length or polydispersity of the
polymers at this stage.
Figure 3. SEC chromatograms of Bi-PAAm(DTPA)-50, Bi-PAAm-
(DTPA)-40, and Bi-PAAm(DTPA)-20 in water at 30 °C containing
0.2 M KNO3, 25 mM phosphate buffer (pH 8.5), and 200 ppm NaN3.
The traces were analyzed by comparison to PMAA standards.
calculated by reference to poly(methacrylic acid) standards are
collected in Table 2. For the shortest chain polymer (DPn =
1
20), it is possible to compare H NMR pendant group signals
to those of peaks from the biotin. In this analysis, the integral of
peak a (1 H) from the biotin (Figure 4) at 4.3 ppm was
compared to the integral of the signal at 1.0−2.25 ppm
associated with the backbone protons d and e. In this way we
determined DPn(NMR) = 20.4 1.4 (Table 2). By comparing
the integrals of peaks at 2.8−3.4 ppm associated with the
protons of the pendant groups and DTPAs with the integrals of
the protons on the backbone, we determined that there are 0.99
DTPA per repeat unit, i.e., quantitative attachment of DTPA
units to each pendant group. The corresponding spectra of Bi-
PAAm(DTPA)-40 and Bi-PAAm(DTPA)-50 are presented in
Figure S9.
While the DPn can be measured by 1H NMR for Bi-
PAAm(DTPA)-20 and estimated for Bi-PAAm(DTPA)-40 and
2838
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Figure 4. 1H NMR spectrum of Bi-PAAm(DTPA)-20 in D2O. DPn = 20.4 1.4 was calculated by comparing the integral for peaks d + e to that of a
(1 H) from the biotin at 4.3 ppm.
Bi-PAAm(DTPA)-50, converting weighed amounts of these
samples to moles of polymers requires knowledge of the
effective molecular weight of the samples. These samples,
obtained by DTPA attachment at pH 8.5, followed by washing
with DI water and lyophilization, contain traces of residual
moisture and sodium ions associated with partial neutralization
of the −COOH groups of the DTPA units. In order to
determine the moisture and Na+ ion content of each sample,
aliquots of each powder were subjected to two-step TGA. As
shown by Majonis et al.,35 heating at 100 °C leads to loss of
moisture without subsequent decomposition of the polymer.
Subsequent heating in air at 600 °C converts all nonvolatile
material to Na2CO3. The TGA traces are presented in Figure
S10 of the Supporting Information. The results of this analysis
and calculated effective Mn values are shown in Table 2.
Complexation of Biotin-MCPs with tmFab-SAv and
Radiolabeling with 111In. While SAv can in principle bind up
to four MCPs, we did not attempt to saturate the binding of
polymer to tmFab-SAv, due to possible interference with the
tertiary structure of the binding epitope of the trastuzumab Fab
fragment for HER2. 111In labeling was conducted under slightly
acidic conditions (100 mM acetate buffer, pH 6.0), as is
routinely performed,40 to inhibit possible hydrolysis of the In3+
ions.
Figure 5. HPLC-SEC chromatogram of tmFab-SAv after treatment
with 1 equiv of Bi-PAAm(DTPA)-40-111In. The narrower peak (green)
is the response of the UV detector at 280 nm. The larger diameter of
the flow cell in the radioactivity detector leads to peak broadening.
[Flow rate, 0.35 mL/min; BioSep-SEC-4000 column (300 mm × 4.6
mm). Eluent 0.1 M NaH2PO4, pH 7.0; injection volume 20 μL; UV
detector at 280 nm.]
with tmFab-SAv (Figure 6). The Bi-MCP-tmFab-SAv complex
eluted about 2 min earlier than tmFab-SAv not complexed to
Bi-MCP. The shift in elution to shorter retention times for a
polymer conjugate is similar to results reported by Zheng et
al.41 This shift is too large to be caused only by the increase of
molecular weight of the Bi-MCP-tmFab-SAv complex com-
pared to the case of tmFab-SAv. Bi-PAAm(DTPA)-20, Bi-
PAAm(DTPA)-40, and Bi-PAAm(DTPA)-50 are negatively
charged polyelectrolytes. In their random conformation state in
100 mM phosphate buffer, these Bi-MCPs may make a
disproportionately large contribution to the hydrodynamic
volume of the Bi-MCP-tmFab-SAv complex, resulting in a
shorter retention time.
The binding of the Bi-MCP to tmFab-SAv after labeling with
111In was examined by HPLC-SEC using UV/vis (280 nm) and
radioactivity detectors. The results for Bi-PAAm(DTPA)-40 are
presented in Figure 5, with the corresponding chromatograms
for Bi-PAAm(DTPA)-20 and Bi-PAAm(DTPA)-50 provided in
Figure S11. The superposition of the UV signal, which detects
the tmFab-SAv, and the radioactivity signal, which detects the
111In bound to the Bi-MCP, confirms that the polymer was
complexed to tmFab-SAv.
In order to verify complexation of Bi-MCPs to tmFab-SAv,
we compared the HPLC-SEC chromatograms using UV
detection only at 280 nm of tmFab-SAv-BiPAAm(DTPA)-20
In Figure 7, HPLC-SEC chromatograms of RICs formed
from the three Bi-MCPs with different DPn values are shown.
Although Bi-MCP-tmFab-SAv complexes with longer polymer
2839
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
Bi-PAAm(DTPA)-50 has the largest polydispersity, and tmFab-
SAv-Bi-PAAm(DTPA)-50 shows the broadest peak in the
HPLC-SEC analysis.
Evaluation of Maximum Achieved Specific Activity of
Bi-MCPs. To examine the maximum SA of the three Bi-MCPs
for complexing 111In, a comparison of the SA achievable was
made with that for tmFab(DTPA)-SAv (Table 3). The
maximum SA achieved was calculated based on the minimum
mass which provided a LE > 80%. The SA values achieved for
all three polymers were very similar (175 2 MBq/μg) with a
LE ≥ 92%. The maximum SA achieved on a per mole basis was
223 20, 429 50, and 560 60 MBq/μmol, respectively, for
Bi-MCPs harboring 20, 40, or 51 pendant DTPA chelators.
These results indicate that the 111In3+ binding capacity increases
linearly with the number of DTPA groups on the polymer.
They further suggest that there are no negative cooperativity
effects that prevent very small amounts of these polymers from
complexing 111In. The maximum SA achieved on a per mole
basis for the different polymers also indicates that SA is
dependent on chain length. Particularly notable is that the
maximum SA achieved with tmFab-SAv modified with two
DTPA chelators per molecule was only 0.3 MBq/μg (3.4 MBq/
μmol), which is 55-fold to 138-fold lower than that for Bi-
MCPs. These results not only confirm Torchilin’s suggestion
that polymers with multiple chelators conjugated to a
bioaffinity agent can achieve a higher per mole SA than one
bearing a small number of these groups,3 but they show for
these three polymers that the SA increases linearly with the
number of DTPA groups, which corresponds to the chain
length.
Figure 6. HPLC-SEC chromatogram of tmFab-SAv-Bi-PAAm
(DTPA)-20-111In and tmFab-SAv. Flow rate, 0.35 mL/min; BioSep-
SEC-4000 column (300 mm × 4.6 mm). Eluent 0.1 M NaH2PO4, pH
7.0; injection volume 20 μL; UV detector at 280 nm. The large shift
between the tmFab-SAv and tmFab-SAv-Bi-PAAm(DTPA)-20 con-
firms the complexation of polymer to tmFab-SAv.
Figure 7. Comparison of HPLC-SEC chromatograms (UV detection)
for tmFab-SAv conjugated to the three different Bi-MCPs of different
chain lengths: tmFab-SAv-Bi-PAAm(DTPA)-20-111In, tmFab-SAv-Bi-
PAAm(DTPA)-40-111In, tmFab-SAv-Bi-PAAm(DTPA)-50-111In. Flow
rate, 0.35 mL/min; BioSep-SEC-4000 column (300 mm × 4.6 mm).
Eluent: 0.1 M NaH2PO4, pH 7.0; injection volume 20 μL; UV detector
at 280 nm.
HER2 Binding. To assess the HER2 binding of 111In-labeled
tmFab-SAv-Bi-MCP complexes, their specific binding to
SKOV-3 human ovarian cancer cells (1 × 106 HER2/cell)
was determined and compared to that of 111In-labeled
tmFab(DTPA)-SAv.
The results of these assays are shown in Figure 8, presented
as percentage of RICs bound per 1.5 × 105 cells. For 111In-
labeled tmFab(DTPA)-SAv, the proportion of specific binding
was (88.8 2.1)%. In comparison, the proportion of specific
binding for 111In-labeled tmFab-SAv-PAAm(DTPA)-20,
tmFab-SAv-PAAm(DTPA)-40, and tmFab-SAv-PAAm-
(DTPA)-50 was (40.93 1.6)%, (26.5 5.4)%, and (26.3
1.4)%, respectively.
chains show slightly earlier retention times, the differences
among these three complexes are not as great as the differences
in retention times between the Bi-MCP-tmFab-SAv complexes
and tmFab-SAv. The differences in peak broadening of the
three Bi-MCP-tmFab-SAv complexes (Figure 7) compared to
tmFab-SAv (Figure 6) are likely due to the differences in
polydispersity of the MCPs (Table 2). Of the three polymers,
Table 3. Maximum Achieved Specific Activity (SA) for 111In Labeling of Metal-Chelating Polymers (MCPs) of Varying Chain
Lengths and tmFab(DTPA)-SAv Which Harbors 2 DTPA Chelators
a
b
complex
mass (μg) amount of DTPA (nmol) labeling efficiency (%) specific activity (MBq/μg) specific activity (MBq/μmol × 104)
Bi-PAAm(DTPA)20
2
3.28
98.6 0.1
97.5 0.1
94.1 1.0
97.9 0.3
97.2 0.1
92.8 1.0
96.3 1.4
98.0 0.2
95.5 1.0
84.4 0.9
1.80 0.001
18.0 0.02
174 1.5
2.33 0.01
23.1 0.3
223 20
0.2
0.02
2
0.328
0.0328
3.28
Bi-PAAm(DTPA)40
Bi-PAAm(DTPA)50
tmFab(DTPA)-SAv
1.80 0.01
18.0 0.01
172 2.0
4.53 0.1
45.0 0.2
429 50
0.2
0.02
2
0.328
0.0328
3.22
1.80 0.03
18.1 0.03
177 1.8
5.65 0.8
57.4 0.9
560 60
0.2
0.02
0.322
0.0322
0.16
10
0.31 0.003
4.05 0.04
a
. Decreasing amounts of Bi-MCP or tmFab(DTPA)-SAv were labeled with a constant amount of 111In (3.7 MBq). Each experiment was done in
b
triplicate; the standard error of the mean is reported. . The labeling efficiency was determined by ITLC and used to calculate the achievable activity
for the polymers.
2840
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
binding to HER2+ SKOV-3 cells was decreased approximately
2-fold. The shortest chain polymer (DPn = 20) yielded
modestly higher specific HER2 binding complexed to tmFab-
SAv than the two longer polymers (DPn = 40, 50), but the
differences were not significant. The maximum achievable SA of
these polymers for complexing 111In was 55- to 138-fold higher
than that for DTPA-tmFab-SAv, and it was linearly correlated
with chain length and the number of pendant DTPA groups.
These polymers provide an attractive route to increasing the
amount of 111In delivered to HER2+ tumor cells per receptor
recognition event, but the effects of polymer composition on
nonspecific binding to cells should be further investigated.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR spectra of monomer 1, of the precursors to the CTA
2, of Bi-PBocNAAm-20 and Bi-PBocNAAm-50, of Bi-
PBocNAAm-20 after end group modification, of the (depro-
tected) polyamino-polymer 5 (DPn = 40) after removal of the
tBOC groups, and of Bi-PAAm(DTPA)-40 and Bi-PAAm-
(DTPA)-50; size exclusion chromatograms of Bi-PBocNAAm-
20, Bi-PBocNAAm-40, and Bi-PBocNAAm-50 at 80 °C in
NMP; TGA traces of Bi-PAAm(DTPA)-n with n = 20, 40, 50;
and HPLC-SEC chromatograms of tmFab-SAv-Bi-PAAm-
(DTPA)-20-111In and tmFab-SAv-Bi-PAAm(DTPA)-50-111In
with UV and radioactivity detection. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 8. Percent binding of RICs bound to HER2+ SKOV-3 human
ovarian cancer cells for 111In-labeled tmFab(DTPA)-SAv and for 111In-
labeled tmFab-SAv-Bi-PAAm(DTPA)-20, tmFab-SAv-Bi-PAAm-
(DTPA)-40, and tmFab-SAv-Bi-PAAm(DTPA)-50. Error bars repre-
sent the mean SEM, n = 3. The asterisk represents p < 0.0001 for
comparison with polymer RICs.
These results revealed that complexation of tmFab-SAv with
Bi-MCPs increased nonspecific binding to SKOV-3 cells (i.e.,
binding that could not be displaced by an excess of unlabeled
trastuzumab IgG) such that the proportion of specific binding
was lower than that found with tmFab(DTPA)-SAv. There was
a modest chain length effect observed for the Bi-MCPs, in that
the polymers with DPn = 40 and 50 exhibited a slightly lower
proportion of HER2 specific binding.
Although there was an increase in the nonspecific binding of
tmFab-SAv complexed to Bi-MCPs, these complexes retained
specific binding to HER2. The reasons for the increase in
nonspecific binding are not currently known and are an area for
further investigation. Possible explanations include steric effects
of the hydrodynamic volume of the polymer and the
electrostatic effects associated with the negative charges along
the polymer backbone. We anticipate that further studies with
polymers of different backbone structure, polymers with
different pendant groups (for example, zwitterionic pendant
groups with no net charge), polymers with a broader range of
chain lengths, and variation of the site of attachment on Fab or
an intact antibody will provide a richer appreciation of these
effects from which we expect to develop mechanistic
explanations of how the immunoreactivity of antibodies is
related to polymer conjugation.
AUTHOR INFORMATION
Corresponding Authors
*raymond.reilly@utoronto.ca (R.M.R.).
*mwinnik@chem.utoronto.ca (M.A.W.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support from the
Canadian Institutes of Health Research/Natural Sciences and
the Engineering Research Council Collaborative Health
Research Program (Grant No. CHRPJ 365423-09; CPG-
95268), as well as from DVS Sciences. Y.L. thanks the Chinese
Scholarship Council for a scholarship to come to the University
of Toronto. We also thank Dr. M. Soleimani for assistance with
the TGA measurements, as well as D. Majonis for helpful
discussions.
ABBREVIATIONS
CONCLUSIONS
■
■
Using RAFT polymerization, we synthesized three well-defined
polyacrylamides of different chain length, all with a biotin end
group. Through postpolymerization modification, metal-chelat-
ing (DTPA) pendant groups were attached to each repeat unit.
The polymers were characterized by a combination of 1H NMR
and SEC to determine DPn and polydispersity, and then by
TGA to determine the moisture and Na+ ion content of the
polymers obtained by lyophilization at pH 8.5. These MCPs
were conjugated to tmFab-SAv through the strong affinity (Ka
= 1015 M−1) between biotin and SAv. After labeling with 111In,
these RICs were evaluated by HPLC-SEC to confirm the
conjugation of MCPs. Cell binding assays revealed that the
tmFab-SAv-MCP complexes have a higher proportion of
nonspecific binding compared to tmFab-SAv modified with
DTPA and labeled with 111In. Thus, the proportion of specific
ACVA, azobis(4-cyanovaleric acid); AMBN, 2,2′-azobis(2-
methylbutyronitrile); BC, breast cancer; Bi-EO2NH2, N-1[-
2,2′-(ethylenedioxy)diethyl-1′-amino]biotinylamide; Bi-MCP,
biotin-end-capped metal-chelating polymer; Bi-PAAm(DTPA),
poly(acrylamide-diethylene-diamide-DTPA; Bi-PFP, biotinyl-
p e n t a fl u o r o p h e n y l e s t e r ; B i s - P F P - A C V , b i s -
(pentafluorophenyl)azobis(4-cyanovalerate); Bi-PBocNAAm,
biotin-poly-(N-{2-[(BOC) aminoethyl]} acrylamide); CTA,
chain transfer agent; CTA-PFP, pentafluorophenyl-[4-(phenyl-
thiocarbonylthio)-4-cyanovalerate]; DCC, N,N′-dicyclohexyl-
carbodiimide; DCM, dichloromethane; DMF, dimethylforma-
mide; DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-meth-
ylmorpholinium chloride; DPn, degree of polymerization; DI,
deionized; DTPA, diethylenetriaminepentaacetic acid; ECD,
extracellular domain; HER2, human epidermal growth factor
2841
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842

Biomacromolecules
Article
(20) Costantini, D. L.; Hu, M.; Reilly, R. M. Cancer Biother.
Radiopharm. 2008, 23, 3−24.
receptor-2; HPLC, high pressure liquid chromatography; ICP-
MS, inductively coupled plasma mass spectrometry; ITLC-SG,
instant thin layer silica gel chromatography; LE, labeling
efficiency; mAb, monoclonal antibody; MCP, metal-chelating
polymer; MWCO, molecular weight cut-off; NHS, N-
hydroxysuccinimide ester; NLS, nuclear localization sequence;
NMP, N-methylpyrrolidone; PBS, phosphate buffered saline;
PEG, polyethylene glycol; PFP, pentafluorophenyl; pfp-OH,
pentafluorophenol; PMMA, poly(methyl methacrylate); RAFT,
reversible addition−fragmentation transfer; RIC, radioimmu-
noconjugate; RBF, round-bottom flask; RT, room temperature;
SA, specific activity; SAv, streptavidin; SEC, size exclusion
chromatography; tBoc-DE, N-(tert-butoxycarbonyl)-1,2-diami-
noethane; TE, triethylamine; TFA, trifluoroacetic acid; TFAA,
trifluoroacetic anhydride; TGA, thermal gravimetric analysis;
THF, tetrahydrofuran; tmFab, trastuzumab Fab fragment;
TOF, time-of-flight
(21) Costantini, D. L.; Bateman, K.; McLarty, K.; Vallis, K. A.; Reilly,
R. M. J. Nucl. Med. 2008, 49, 1498−1505.
(22) Boyer, B.; Bulmus, V.; Davis, T. P.; Ladmiral, V.; Liu, J.; Perrier,
S. Chem. Rev. 2009, 109, 5402−5436.
(23) York, A. W.; Huang, F.; McCormick, C. L. Biomacromolecules
2010, 11, 505−514.
(24) Heredia, K. L.; Nguyen, T. H.; Chang, C. W.; Bulmus, V.; Davis,
T. P.; Maynard, H. D. Chem. Commun. (Cambridge, U. K.) 2008, 28,
3245−3247.
(25) Jia, Z.; Wong, L.; Davis, T. P.; Bulmus, V. Biomacromolecules
2008, 9, 3106−3113.
(26) Pan, H. Z.; Sima, M.; Kopeckova, P.; Wu, K.; Gao, S. Q.; Liu, J.
H.; Wang, D.; Miller, S. C. Mol. Pharmaceutics 2008, 5, 548−558.
(27) Rossi, N. A. A.; Mustafa, I.; Jackson, J. K.; Burt, H. M.; Horte, S.
A.; Scott, M. D.; Kizhakkedathu, J. N. Biomaterials 2009, 30, 638−648.
(28) Allmeroth, M.; Moderegger, D.; Biesalski, B.; Koynov, K.;
Rosch, F.; Thews, O.; Zentel, R. Biomacromolecules 2011, 12, 2841−
̈
2849.
(29) Boyle, A. J.; Liu, P.; Lu, Y.; Weinrich, D.; Scollard, A. D.;
Mbong, G. N. N.; Winnik, M. A.; Reilly, R. M. Pharm. Res. 2012, in
press.
REFERENCES
■
(1) (a) Erdogan, S.; Roby, A.; Sawant, R.; Hurley, J.; Torchilin, V. P.
J. Liposome Res. 2006, 16, 45−55. (b) Weissig, V.; Babich, J.; Torchilin,
V. P. Colloids Surf., B 2000, 18, 293−299. (c) Erdogan, S.; Medarova,
Z. O.; Roby, A.; Moore, A.; Torchilin, V. P. J. Magn. Reson. Imaging
2008, 574−580. (d) Torchilin, V. P. In Handbook of Targeted Delivery
of Imaging Agents; CRC Press: Boca Raton, FL, 1995; pp 149−173.
(e) Stealth Liposomes; Lasic, D., Eds.; CRC Press: Boca Raton, FL,
1995.
(30) Li, B.; Majonis, D.; Liu, P.; Winnik, M. A. Can. J. Chem. 2011,
89, 317−325.
(31) Korshun, V. A.; Pestov, N. B.; Nozhevnikova, E. V.;
Prokhorenko, I. A.; Gontarev, S. V.; Berlin, Y. A. Synth. Commun.
1996, 26, 2531−2547.
(32) Reizelman, A.; Wigchert, S. C. M.; del-Bianco, C.; Zwanaberg, B.
Org. Biomol. Chem. 2003, 1, 950−959.
(2) Torchilin, V. P.; Klibanov, A. L. Crit. Rev. Ther. Drug Carrier Syst.
(33) Roth, P. J.; Wiss, K. T.; Zentel, R.; Theato, P. Macromolecules
2008, 41, 8513−8519.
1991, 7, 275−308.
(3) Torchilin, V. P. Bioconjugate Chem. 1999, 10, 146−149.
(4) Patel, K. Mater. Matters 2010, 5, 1−29.
(5) Grubbs, R. H.; Tumas, W. Science 1989, 243, 907−915.
(6) (a) Hernandez, J. R.; Klok, H.-A. J. Polym. Sci., Part A: Polym.
Chem. 2003, 41, 1167−1187. (b) Hadjichristidis, N.; Iatrou, H.;
Pitsikalis, M.; Sakellariou, G. Chem. Rev. 2009, 109, 5528−5578.
(7) Carlise, J. R.; Weck, M. J. Polym. Sci., Part A: Polym. Chem. 2004,
42, 2973−2984.
(8) Shunmgam, R.; Gaberiel, G. J.; Aamer, K. A.; Tew, G. N.
Macromol. Rapid Commun. 2010, 31, 784−793.
(9) Shunmgam, R.; Tew, G. N. Macromol. Rapid Commun. 2008, 29,
1355−1362.
(34) Bathfield, M.; D’Agosto, F.; Spitz, R.; Charreyre, M.-T.; Delair,
T. J. Am. Chem. Soc. 2006, 128, 2546−2547.
(35) Majonis, D.; Ornatsky, O.; Kinach, R.; Winnik, M. A.
Biomacromolecules 2011, 12, 3997−4010.
(36) Green, M.; Berman, J. Tetrahedron Lett. 1990, 31, 5851−5852.
(37) You, Y. Z.; Manickam, D. S.; Zhou, Q. H.; Oupicky, D.
Biomacromolecules 2007, 8, 2038−2044.
(38) Qiu, X.-P.; Tanaka, F.; Winnik, F. M. Macromolecules 2007, 40,
7069−7071.
(39) Thompson, K.; Michielsen, S. J. Polym. Sci., Part A: Polym. Chem.
2006, 44, 126−136.
(40) Reilly, R. M. In Handbook of Biopharmaceutical Technology, Gad,
S. C., Eds.; John Wiley & Sons, Ltd: Toronto, ON, 2007; pp 987−
1053.
(10) (a) Calzia, K. J.; Tew, G. N. Macromolecules 2002, 35, 6090−
6093. (b) Schubert, U. S.; Hofmeier, H. Macromol. Rapid Commun.
2002, 23, 561−566.
(41) Zheng, C. Y.; Ma, G.; Su, Z. Electrophoresis 2007, 28, 2801−
2807.
(11) El-Ghayoury, A.; Hofmeier, H.; de Ruiter, B.; Schubert, U. S.
Macromolecules 2003, 36, 3955−3959.
(12) Aamer, K. A.; Tew, G. N. Macromolecules 2004, 37, 1990−1993.
(13) Shunmugam, R.; Tew, G. N. J. Am. Chem. Soc. 2005, 127,
13567−13572.
(14) Shunmugam, R.; Tew, G. N. Chem.Eur. J. 2008, 14, 5409−
5412.
(15) Bandura, D. R.; Baranov, V. I.; Ornatsky, O. I.; Antonov, A.;
Kinach, R.; Lou, X.; Pavlov, S.; Vorobiev, S.; Dick, J. E.; Tanner, S. D.
Anal. Chem. 2009, 81, 6813−6822.
(16) Lou, X.; Zhang, G.; Herrera, I.; Kinach, R.; Ornatsky, O.;
Baranov, V.; Nitz, M.; Winnik, M. A. Angew. Chem., Int. Ed. 2007, 46,
6111−6114.
(17) Majonis, D.; Herrera, I.; Ornatsky, O.; Shulze, M.; Lou, X.;
Soleimani, M.; Nitz, M.; Winnik, M. A. Anal. Chem. 2010, 82, 8961−
8969.
(18) Bendall, S. C.; Simonds, E. F.; Qiu, P.; Amir, E. D.; Krutzik, P.
O.; Finck, R.; Bruggner, R. V.; Melamed, R.; Trejo, A.; Ornatsky, O. I.;
Balderas, R. S.; Plevritis, S. K.; Sachs, K; Pe’er, D.; Tanner, S. D.;
Nolan, G. P. Science 2011, 332, 687−696.
(19) Costantini, D. L.; Chan, C.; Cai, Z.; Vallis, K. A.; Reilly, R. M. J.
Nucl. Med. 2007, 48, 1357−1368.
2842
dx.doi.org/10.1021/bm300843u | Biomacromolecules 2012, 13, 2831−2842