Intracellular routing in breast cancer cells of streptavidin-conjugated trastuzumab fab fragments linked to biotinylated doxorubicin-functionalized metal chelating polymers

Article abstract of DOI:10.1021/bm401483a

We describe the synthesis of a heterotelechelic metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree of polymerization DP_n = 50 (PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic acid (DTPA) pendant groups as the binding sites for metal ions. We compared its behavior in cell-uptake experiments with a similar polymer (Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab (tmFab) fragments covalently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2 ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with ¹¹¹In (an Auger electron emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In³⁺ and visualized their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with ¹¹¹In. Both radiolabeled complexes showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in human breast cancer cells expressing HER2. Further study is needed to understand the impact of polymer charge on cellular uptake and distribution to intracellular compartments.

Full text of DOI:10.1021/bm401483a

Article
pubs.acs.org/Biomac
Intracellular Routing in Breast Cancer Cells of Streptavidin-
Conjugated Trastuzumab Fab Fragments Linked to Biotinylated
Doxorubicin-Functionalized Metal Chelating Polymers
Peng Liu,† Zhongli Cai,‡ Jae W. Kang,† Amanda J. Boyle,‡ Jarret Adams,§ Yijie Lu,†
Ghislaine Ngo Ndjock Mbong,‡ Sachdev Sidhu,§ Raymond M. Reilly,*,‡,∥ and Mitchell A. Winnik*,†
†Department of Chemistry, University of Toronto, 80 St. 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
§Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario,
Canada M5S 3E1
S
* Supporting Information
ABSTRACT: We describe the synthesis of a heterotelechelic
metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide
with a number average degree of polymerization DPn = 50
(PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as
functional chain ends and diethylenetriaminepentaacetic acid
(DTPA) pendant groups as the binding sites for metal ions.
We compared its behavior in cell-uptake experiments with a
similar polymer (Bi-MCP) without Dox. These MCPs were
complexed with trastuzumab Fab (tmFab) fragments cova-
lently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi
and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast
cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that
incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2
ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with 111In (an Auger electron
emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted
intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-
MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In3+ and visualized
their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell
fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with 111In. Both radiolabeled complexes
showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to
encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in
human breast cancer cells expressing HER2. Further study is needed to understand the impact of polymer charge on cellular
uptake and distribution to intracellular compartments.
normal cells except cardiac myocytes, numerous studies have
focused on immunotherapy of HER2-positive tumors.8
INTRODUCTION
■
HER2 is the second member of the human epidermal growth
factor receptor (EGFR) family, and is encoded by the HER2/
neu proto-oncogene.1 Overexpression of HER2 is associated
with a wide spectrum of cancers, including lung, breast, and
ovarian, as well as adenocarcinomas of the colon and salivary
gland.2−4 In 1987, Slamon et al. reported that overexpression of
HER2 was found in about 30% of 189 primary breast
carcinomas and was correlated with a poor prognosis.5
Moreover, overexpression of HER2 may contribute to the
transformation of normal cells into a neoplastic phenotype.6,7
Because of its ready accessibility as a transmembrane
glycoprotein and the limited expression of HER2 in most
For more than 20 years, the humanized monoclonal antibody
trastuzumab (Herceptin) has been widely used for the
treatment of HER2-positive metastatic breast cancer.9 The
mechanism of action of trastuzumab is not completely
understood, but this agent combined with chemotherapy
(e.g., paclitaxel or docetaxel) is now the standard-of-care for
this type of breast cancer.10 Nonetheless, only half of the
patients with HER2-positive metastatic breast cancer benefit
Received: October 3, 2013
Revised: January 24, 2014
Published: February 7, 2014
© 2014 American Chemical Society
715
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
from trastuzumab-chemotherapy combinations, and almost all
patients who initially respond to this treatment acquire
resistance within a year.10
polymers could be rapidly bound to tmFab-SAv and then
labeled with 111In. One type of polymer, with a polyacrylamide
backbone, was synthesized by reversible addition−fragmenta-
tion transfer (RAFT) polymerization.18 Polypeptide polymers
with a polyaspartamide backbone or a polyglutamide backbone
were synthesized by ring-opening polymerization of the
corresponding benzylaspartate N-carboxyanhydride (NCA)
and benzylglutamate NCA.19 Following construction and
characterization of the polymer backbone, the DTPA groups
were introduced by a sequence of pendant group trans-
formations that maintained the overall length of the polymer. In
this way, polymers with a number average degree of
polymerization (DPn) of 20 to 50 monomer units per polymer
were obtained, with polydispersities (PDI) in the range of 1.1
to 1.2.
Several strategies to increase the effectiveness of trastuzumab
therapy for HER2-positive tumors have been reported. These
approaches couple HER2 targeting using trastuzumab with the
delivery of cytotoxic agents to tumor cells.11−14 For example,
trastuzumab has been chemically modified with an anthracy-
cline12 (which binds to DNA) and auristatin13 (that disrupts
microtubules) to improve the therapeutic index, or with
mytansinoid (DM1), which also targets microtubules. The
conjugate trastuzumab-DM1 has proven to be very effective for
treatment of HER2-positive breast cancer, including tumors
that have developed resistance to treatment with trastuzumab-
containing regimens.14
Another emerging approach is to generate radioimmuno-
conjugates (RICs) by labeling trastuzumab with cytotoxic
radionuclides. Costantini, et al.15 derivatized trastuzumab with
DTPA as a carrier of 111Indium (111In), which emits Auger
electrons in addition to its γ-emissions. These electrons have
low energy (<25 keV) and deposit this energy over nanometer-
micrometer distances resulting in high linear energy transfer,
which causes nuclear DNA double strand breaks. However,
because of the ultrashort range of these electrons, 111In is most
effective for causing lethal DNA breaks if it is in close proximity
to the cell nucleus. For this reason, Costantini et al. also
derivatized trastuzumab with multiple copies of a nuclear-
localizing sequence (NLS) peptide. The NLS peptides are
believed to interact with importins α/β, which are intracellular
transport proteins that enable translocation across the nuclear
pore complex. These authors found that 111In-NLS-trastuzumab
was 8-fold more potent on a concentration basis at decreasing
the clonogenic survival of HER2 amplified SK-BR-3 cells with
high HER2 density (2 × 106 per cell) than unlabeled
trastuzumab.
In a recent study, we examined the influence of MCP
structure on the tumor and normal tissue biodistribution of
trastuzumab in a mouse model, particularly the effects of the
charge and backbone composition of the polymers. These
experiments involved Bi-MCPs with polyacrylamide [Bi-
PAAm(DTPA40)] and polyglutamide [Bi-PGlu(DTPA28)] or
polyaspartamide [Bi-PAsp(DTPA)33] backbones linked to SAv-
tmFab and labeled with 111In.17 The effect of saturation of
uncomplexed DTPA groups with stable In3+ ions was also
examined. Biodistribution was assessed by microSPECT/CT
imaging and by ex vivo γ-scintillation counting of tissues by
taking advantage of the two γ-photons emitted by 111In. Our
results showed that MCPs with a zwitterionic polyglutamide
backbone were the most suitable for constructing polymer-
tmFab immunoconjugates, since these exhibited the lowest
accumulation in the liver and preserved the specific tumor
accumulation of tmFab in HER2-overexpressing SKOV-3
human ovarian cancer xenografts in athymic mice.17 High
liver uptake was observed for MCPs with a polyacrylamide
backbone, an effect that we attributed to the negative charges
associated with the repeat unit of this polymer. Saturating
uncomplexed DTPA groups with In3+lowered the net charge
per repeat unit of the polymer and decreased the liver uptake of
the RIC. The maximum specific activity for labeling with 111In
was increased by more than 100-fold for the MCPs compared
to tmFab conjugated to two DTPA per molecule.18
In this paper we describe cell uptake experiments with
HER2-overexpressing SK-BR-3 cells with a pair of tmFab-SAv-
Bi-MCP complexes in which we compared two carefully
matched MCPs. One was a novel heterotelechelic polyacryla-
mide MCP that contained a biotin group at the initiating end to
permit linkage to tmFab-SAv, and a covalently bound
doxorubicin (Dox) at the other end. The other was biotin-
polyacrylamide of very similar length and composition, lacking
the terminal Dox. Both were synthesized by RAFT polymer-
ization. Doxorubicin is a widely used anticancer drug.20 Dox
interacts with proteosomes and is subsequently transferred to
the cell nucleus, where it intercalates into DNA, inhibiting
DNA replication, leading to apoptosis.20 Dox has been found to
be a radiosensitizer for the DNA-damaging effects of the Auger
electrons emitted by 111In, possibly by inhibiting DNA repair.21
In addition, a related analog, daunorubicin labeled with the
Auger electron-emitter 125I, has been shown to directly cause
DNA strand breaks in SK-BR-3 cells.22
One limitation of 111In-NLS-trastuzumab was its low specific
activity (SA). In the example referred to above, the SA achieved
indicated that only 1 in 50 antibodies were labeled with 111In.15
Thus, in the clonogenic assay experiments, a large proportion of
the targeted HER2 was bound by nonradiolabeled antibodies.
This problem can in principle be overcome by attaching a metal
chelating polymer (MCP)16 with multiple sites for complexing
111In to trastuzumab. In this way, the probability that each
antibody carried one or more radionuclides would be
substantially enhanced.
Recently we described a facile approach to append MCPs,
each carrying multiple diethylenetriaminepentaacetic acid
(DTPA) groups for complexing 111In, onto Fab fragments of
trastuzumab (tmFab) with the idea of amplifying the
radiometal ion delivery per HER2 recognition event.17−19
tmFab was first linked covalently to streptavidin (SAv) through
a polyethylene glycol (PEG24) spacer. A series of biotinylated
MCPs (Bi-MCP), each bearing a terminal biotin, was
synthesized for noncovalent but high affinity (Ka = 1015 M−1)
binding to the SAv moiety. Our goal was to examine a family of
MCPs that differed in backbone structure (polyacrylamide,
polypeptide), chain length, and pendant groups, with the goal
of exploring how these factors affected biodistribution in vivo
and cell uptake in vitro.
These polymers were synthesized by “living” polymerization
reactions that allowed control over chain length and end group
functionality and yielded polymers with narrow length
distributions. Because of the terminal biotin group, these
In this study, we hypothesized that Dox could act as a nuclear
localization moiety for MCPs to deliver 111In into close
proximity to DNA to maximize its lethal DNA-damaging
effects. From a practical point of view, Dox has an optical
716
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
Scheme 1. Synthesis Route for Bi-DTPA and Dox-Maleimide (A) and for Biotin Containing RAFT Polymer (B)
a
Scheme 2. Synthesis Route for Bi-PAAm(DTPA)50-Dox and the Chemical Structure of Bi-PAAm(DTPA)50
a
These two polymers have the same backbone structure and biotin end groups, as well as almost identical chain lengths.
absorption band in the visible region of the spectrum (λab = 480
nm), and its strong characteristic fluorescence (λem = 550 nm)
enables visualization of its intracellular distribution by confocal
microscopy. In cell fractionation studies, we compared the
behavior of tmFab-SAv-Bi-MCP-Dox with tmFab-SAv-Bi-MCP,
the essentially identical MCP lacking the Dox end group. In
these experiments, tmFab-SAvs were complexed with MCPs
that were labeled with 111In. Their intracellular routing was
determined by measuring the radioactivities of isolated cell
compartments. In addition, we used surface plasmon resonance
(SPR) measurements employing the extracellular domain
(ECD) of HER2 to determine the effect of linking of Bi-
717
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
polymer (9.2 mg) (Bi-PAAm(DTPA)50Dox, which we will also refer to
as “Bi-MCP-Dox”.
MCP and Bi-MCP-Dox to tmFab-SAv on the binding affinity of
the Fab fragments.
Breast Cancer Cells. HER2+ SK-BR-3 cells were purchased from
the American Type Culture Collection (Manassas, VA, U.S.A.) and
cultured in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO,
U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco-
Invitrogen Inc., Carlsbad, CA, U.S.A.) and 1% penicillin-streptomycin
(P/S, Gibco-Invitrogen Inc.). A nuclei isolation kit (Nuclei EZ Prep
Nuclei Isolation Kit, NUC-101; Sigma-Aldrich) was used to separate
the cell nuclei from the cytoplasm. The 6-well cell plates were
purchased from Sarstedt. In the cell staining experiments we used
Hoechst 33342 dye (Molecular Probes, Inc.) to stain cell nuclei and
wheat germ agglutinin-Alexa Fluor 594 (WGA-Alexa) to stain cell
membranes (Molecular Probes, Inc.). The buffers for the In3+ labeling
were treated with Chelex-100 resin (Biorad) to remove cationic trace
metals.
Preparation of Immunoconjugates Containing tmFab-SAv.
Fab fragments of trastuzumab (tmFab) and streptavidin-conjugated
tmFab (tmFab-SAv) were prepared as reported by Boyle et al.17 To
form complexes of tmFab-SAv with biotin-functionalized MCPs and
with Bi-DTPA, solutions of the two components were prepared in PBS
at 1 mg/mL. The conjugate was prepared by mixing tmFab-SAv with
Bi-MCPs in a 1:1 mol ratio and stirring for 1 h at room temperature.
tmFab-SAv and Bi-DTPA were mixed in a mole ratio of 1:3.
Preparation of Immunoconjugates for Confocal Fluorescence
Microscopy. PBS containing InCl3 (stable indium) solution in sodium
acetate (NaOAc) buffer (100 mM, pH 6.0, 8.6 μL) was added to the
tmFab-SAv-Bi-MCP-Dox (11 μg polymer and 37 μg tmFab-SAv) in
PBS prepared as described above. The mixture was kept at room
temperature for 1 h. In order to remove excess In3+, the solution was
subsequently transferred to a 30 kDa MWCO spin-filter, diluted with
NaOAc buffer and washed with several passages of NaOAc buffers (10
× 400 μL, pH 6.0, 100 mM). The residual solution in the upper
chamber of the spin-filter was then diluted with PBS buffer (3 mL, pH
7.4) and collected.
Preparation of RICs for Cell Fractionation Studies. Two cell
fractionation experiments were conducted. In the first experiment,
tmFab-SAv (7.5 μg) complexed with Bi-MCP (2.2 μg), Bi-MCP-Dox
(2.2 μg), Bi-DTPA (0.2 μg) were compared. The RICs were
radiolabeled with 111InCl3 in NaOAc buffer (pH 6.0) to a specific
activity of 0.25 MBq/μg at RT for 1 h. In the second experiment, two
samples of tmFab-SAv-Bi-MCP−111In were prepared. One was used
directly. The other RIC was treated for 1 h with an excess of stable
InCl3 solution (3.39 μL, 3 mg/mL) to saturate the In3+ binding sites
on the polymers. The RICs were purified from free In3+ by spin-filter
tubes (50 kDa MWCO). The labeling efficiency of the three samples
was measured by instant thin layer silica gel chromatography (ITLC-
SG) developed in 100 mM sodium citrate, pH 5.0. The labeling
efficiency of tmFab-SAv-Bi-MCP-Dox was 99.6% and that of tmFab-
SAv-Bi-MCP was 99.5%. The labeling efficiency of tmFab-SAv-Bi-
DTPA was 79%. After one-time purification by spin-filter (50 kDa
MWCO, 1.5 × 104 × g, 10 min), its radiochemical purity increased to
97%.
Surface Plasmon Resonance (SPR) Measurements. The ECD
of HER2 (R&D Systems, Minnesota, U.S.A.) in 50 mM phosphate
buffer (pH 4.5) was immobilized onto an EDAC/sulfo-NHS activated
sensor chip surface at 30 μL/min for 5 min in the vertical direction.
Immobilization levels were monitored to ensure approximately 500
response units were immobilized; a second injection was used if
needed. Immobilized ECDs were then stabilized with PBS for 30 s and
0.85% H3PO4 for 18 s, each at 100 μL/min. For the Fab binding
assays, the highest concentration used for tmFab-SAv was 246 nM, and
this solution was further diluted with PBS containing 0.05% Tween 20
in 2-fold series to produce a series of five concentrations ranging from
246 to 15.4 nM. Binding measurements of tmFab-SAv-Bi-MCP and
tmFab-SAv-Bi-MCP-Dox were initiated with a concentration of 122
nM, followed by serial dilution to obtain five concentrations ranging
from 122 to 7.6 nM. All the binding assays were carried out at 25 °C at
a flow rate of 100 μL/min for 1 min. The dissociation steps were
monitored for 10 min. The kinetic parameters were calculated by
Our most important result was that the MCP itself strongly
promoted cell uptake and delivery to the cell nucleus. The
polymer with the terminal Dox led to somewhat lower nuclear
uptake. These findings have important implications for the
future development of MCPs as carriers of 111In in radio-
immunoconjugates intended for Auger electron radioimmuno-
therapy.
EXPERIMENTAL SECTION
■
Instrumentation. HPLC and SEC. Size exclusion chromatography
(SEC) measurements of precursor polymers were conducted with N-
methylpyrrolidone (NMP) containing 0.2 wt % LiCl as the eluent at
80 °C. Water-soluble MCPs were examined by aqueous SEC with an
eluent of phosphate buffer (pH 8.5, 25 mM, containing 0.2 M KNO3
and 200 ppm NaN3). High pressure liquid chromatography (HPLC)
measurements of samples containing tmFab or 111In were conducted
using 100 mM NaH2PO4 buffer, pH 7.0, as eluent. Measurements of
small organic molecules were conducted by HPLC with mass
spectrometry detection (HPLC-MS) using a C18 column. The
gradient consisted of eluents A (water with 1% formic acid) and B
(acetonitrile with 1% formic acid). Details are provided in the
Supporting Information (SI).
Other Instrumentation. 1H NMR measurements employed a
Varian 400 spectrometer operating at 400 MHz. The γ-counter used
for radioactivity measurements was a 1480 Wizard 3 (Perkin Elmer,
Turku, Finland). ESI mass analyses were performed on QStar model
quadrupole time-of-flight mass spectrometer (AB Sciex, Concord,
ON). MALDI-TOF spectra were obtained on Waters MICROMASS
MALDI micro MX Mass Spectrometer. The reflectron mode was
employed for measurements of doxorubicin-maleimide (Dox-Malei-
mide) with dithranol as the matrix. Confocal fluorescence images were
obtained with a Zeiss LSM 510 confocal microscope with a Plan-
Apochromat 6.3×/1.2 water objective and laser scanning set to z-stack
mode. The fluorescence excitation laser wavelengths were set to 555,
405, and 488 nm, respectively, to excite three different dyes. Surface
plasmon resonance (SPR) measurements were performed on a
ProteOn XPR36 Protein Interaction Array System (BioRad).
Materials. The biotin-end-capped metal chelating polymer Biotin-
poly(3,6,9-triscarboxymethyl-3,6,9-triazaundecandioicacid-amido-N-
ethylamido acrylamide) [Bi-PAAm(DTPA)50], with a mean degree of
polymerization of DPn ≈ 50, was previously reported in ref 18. For
simplicity, this MCP is referred to as “Bi-MCP”. The synthesis and
characterization of 3,6,9-triscarboxymethyl-3,6,9-triazaundecandioica-
cid-amido-3,6-dioxaoctane-1,8-biotinamide (Monobiotin-DTPA, Bi-
DTPA) as well as doxorubicin-maleimide (Dox-Maleimide; see
Scheme 1) are described in the SI.
Synthesis of a Heterotelechelic MCP with a Terminal
Doxorubicin. Polymer synthesis began with the RAFT polymer-
ization of N-{2-[(BOC) aminoethyl]} acrylamide (BocNAAm) with a
biotin-containing chain transfer agent as described in ref 18. Through a
series of postpolymerization modifications, the dimeric polymer Bi-
PAAm(DTPA)50-disulfide (polymer 4, Scheme 2) was synthesized and
served as a precursor to attach a terminal Dox. The details of the
synthesis and characterization are given in the SI.
Reduction of the Disulfide and Attachment of Dox to the
Polymer. A solution of TCEP (0.5 M) was freshly prepared in HEPES
buffer (20 mM). Bi-PAAm(DTPA)50-disulfide (10 mg) was dissolved
in HEPES buffer (2 mL). An aliquot of the TCEP solution (0.1 mL)
was added, and the mixture was stirred for 4 h at 40 °C. Dox-
maleimide (1.51 mg, 1.98 mmol) was dissolved in DMF (8 mL); this
solution was added to the TCEP-treated MCP solution, and the
mixture was stirred overnight at 40 °C. The solution was diluted with
ethanol/water (1:1 v/v, 13 mL), transferred to a 15 mL 3kDA MWCO
Millipore Amicon spin filter, and washed with the ethanol/water
mixture (5 × 11 mL), followed by DI water (5 × 11 mL). Finally, the
aqueous solution was freeze-dried to yield the Dox-end-labeled DTPA
718
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
Figure 1. (A) Comparison of SEC chromatograms in NMP (with 0.2 wt % LiCl) at 80 °C of the RAFT polymer 1 (dashed line) and polymeric
dimer 2 (solid line) formed through the disulfide bond linkage. The chromatogram demonstrates the earlier elution time of the polymeric dimer in
comparison with the RAFT polymer due to its larger molecular weight. The traces were analyzed by comparing with PMAA standards. (B) 1H NMR
of Bi-PAAm(DTPA)51-disulfide 3. The integration of peaks from the polymer shows that about 97% of the pendant groups were functionalized with
DTPA. (C) Comparison of SEC traces of Bi-MCP-disulfide 3 and the polymer after TCEP reduction 4 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. The thicker line refers to the
disulfide polymer and the thinner line represents the polymers after TCEP reduction. The retention volume shift and the narrower polydispersity of
the TCEP-treated polymer imply the reduction of the disulfide bond. (D) HPLC chromatogram (BioSep-SEC-4000 column, 300 × 4.6 mm) of
radiolabeled Bi-MCP-Dox monitored at 480 nm and with a radioactivity detector. The eluent was 0.1 M NaH2PO4 at pH 7.0 at a flow rate of 0.35
mL/min. The MCP (0.5 μg/μL in PBS buffer) was labeled with 111In (2 MBq) and 20 μL was injected; UV−vis detector was set at 480 nm. The
broader peak from the radioactivity signal is due to the larger flow cell for this detector.
ProteOn manager software (Bio-Rad Laboratories Ltd. Hercules, CA,
U.S.A.). Analysis was conducted using the kinetic Langmuir model.23
Confocal Fluorescence Microscopy. SK-BR-3 cells in RPMI
1640 medium supplemented with 10% FBS were seeded on coverslips
(12 mm diameter) in 24-well plates. Three groups of experiments were
conducted. In the first, the cells were cocultured with tmFab-SAv-Bi-
MCP-Dox. Before adding the immunoconjugates to the cells, the
concentration of Dox in the solutions was measured by UV−vis at 480
nm. In the second group, cells were cocultured with free Dox at the
same concentration of Dox as the first group. In the third group of
experiments, the cells were treated for 10 min with a 100-fold excess of
trastuzumab compared to the tmFab-SAv-Bi-MCP-Dox complex to
saturate HER2 receptors on the cell surface and then cocultured with
tmFab-SAv-Bi-MCP-Dox to study the nonspecific adsorption of
tmFab-SAv-Bi-MCP-Dox to the cells. All experiments were performed
in triplicate. After incubation at 37 °C overnight, the cells were rinsed
with PBS and incubated with Hank’s Balanced Salt Solution (HBSS)
containing WGA-Alexafluor immunoconjugates (1 μg/mL) and
Hoechst 333324 (4 μg/mL) at 37 °C for 10 min to stain the cell
membranes and nuclei, respectively, and then fixed with 2%
paraformaldehyde PBS solution. Accumulation of the tmFab-SAv-Bi-
MCP-Dox complex within the cells, based on Dox fluorescence, was
observed by confocal fluorescence microscopy in the z-stack mode
with Dox excitation at 488 nm, and detection in the range 490 to 537
nm. Fluorescence excitation for Alexa Fluor 594 was 555 nm with
emission detected at 569 to 679 nm, and for Hoechst 333324,
excitation was 405 nm, with emission detected at 400 to 550 nm.
Cell Fractionation Studies. Subconfluent SK-BR-3 cells were
harvested by trypsinization and seeded into 6-well tissue culture plates
(5 × 105 cells and 1 mL medium per well). The cells were then
cultured for 24 h. After culturing overnight, the cells were treated with
RICs (tmFab-SAv-Bi-MCP−111In, tmFab-SAv-Bi-MCP-Dox-111In and
tmFab-SAv-Bi-DTPA-111In) for another 16 h in the presence of serum-
free medium. The concentration of Fab in each well was 10 nM. After
incubating the cells with RICs for 16 h, the cells were first rinsed with
PBS to remove unbound RICs. Then the cells attached to the plates
were treated with 200 mM NaOAc in 500 mM NaCl, pH 2.5 to
remove cell surface-bound radioactivity, followed by rinsing with PBS.
Both the acidic solution and the rinsing PBS solution were collected
and counted in a gamma counter. Immediately after, cell lysis buffer
was added into the wells, and the cell culture plates were put on ice for
40 min. Cell scrapers were then employed to transfer the lysed cells
into centrifuge tubes. After centrifugation, the supernatant was
collected, and the radioactivity in this fraction representing
internalized RICs in the cytoplasm was counted in a γ-counter. The
nuclear pellet was then sequentially treated with buffers consisting of
(i) 0.2 M sucrose/3 mM CaCl2/ 50 mM Tris Cl, pH 7.6, (ii) 0.14 M
NaCl/10 mM Tris Cl, pH 8.3, and (iii) 1 mM Tris Cl, pH 7.9. Cell
nuclei and chromatin (DNA) were isolated from cells following the
method reported by Reilly et al.24 After each buffer treatment, the
solutions were centrifuged, and the supernatants were collected and
combined. Chromatin was collected as the pellet after the last
centrifugation cycle.
In the second group, cell fractionation experiments with tmFab-
SAv-Bi-MCP−111In were compared with experiments that employed
tmFab-SAv-Bi-MCP−111In saturated with excess stable In3+. Cells were
cultured and treated with RICs in the same manner described above.
Experiments for both groups, In3+-saturated RICs and the RICs
without stable In3+, were conducted in triplicate. In these experiments
the nuclear radioactivity was measured without further fractionation.
719
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
with both radioactivity and UV−vis detectors (Figure 1D). The
overlap of the UV and radioactivity peaks indicates that the
polymer contained both metal-chelating groups (DTPA) and
Dox.
RESULTS AND DISCUSSION
■
Synthesis of the Biotinylated MCP with a Terminal
Dox. MCPs with a biotin at one end were synthesized by
RAFT polymerization of N-{2-[(BOC) aminoethyl]} acryl-
amide (BocNAAm), as previously described,18 using a biotin-
containing chain transfer agent (Scheme 1B). Following the
approach described by Majonis et al.,25 the RAFT end group
was removed by aminolysis, and then the terminal thiol groups
were oxidized to yield dimeric disulfide-coupled polymers. The
disulfide groups protect the terminal thiol groups during
pendant group modification of the polymer. Due to the
sensitivity of Dox to the reaction conditions employed for
pendant group transformation of the initially prepared RAFT
polymer, the strategy was designed to allow for attachment of
Dox to the chain terminus in the last step of the synthesis. The
disulfide bonds of the MCPs were reduced to thiols with
TCEP, followed by reaction with a Dox derivative containing a
maleimide substituent.
The mean degree of polymerization (DPn) of the Bi-
PBocNAAm polymer was characterized by 1H NMR end group
analysis (Figure S1), comparing the integration of the peaks of
the dithiobenzoate protons at 7.91, 7.57, and 7.39 ppm to the
integral of signals of the peaks ranging from 2.59 to 0.38 due to
the polymer backbone protons and the tert-butyl groups. For
this polymer we obtained DPn = 50. Based on previous
experience with this type of polymer,18,25 we assumed that DPn
did not change significantly during the pendant group
transformations.
The Bi-PAAm(DTPA)50Dox polymer (in the fully proto-
nated state) has Mn = 23 kDa. In buffer solutions near neutral
pH, the carboxylic acid groups of the polymers are partially
dissociated. Previous experiments in our laboratory used
thermal gravimetric analysis to characterize the state of
dissociation of the carboxyl groups of a very similar MCP
with pendant DTPA groups.25 For this sample, neutralized at
pH 8.5, washed extensively with DI water and then dried, there
were on average about 2.2 Na+ ions for each DTPA repeat unit.
This number varies with pH and also with the extent of indium
binding.
Complexation of MCPs with tmFab-SAv. Trastuzumab
Fab fragments (tmFab) were covalently linked to streptavidin
(SAv) with a poly(ethylene glycol) (PEG24) spacer (tmFab-
SAv) as described in ref 18. To complex MCPs to tmFab-SAv,
the polymer and protein complex in a 1:1 molar ratio were
incubated in PBS at pH 7.4 at RT for 1 h. An SEC-HPLC
chromatogram of the complex is shown in Figure S8. In the
SPR experiments described below, as well as in the confocal
fluorescence microscopy and cell fractionation studies, the
tmFab-SAv-Bi-MCP complexes were all prepared in the same
manner.
HER2 Recognition. Our design of the RICs is based on the
hypothesis that the Fab fragment of trastuzumab will retain its
targeting ability toward HER2 after complexation with MCPs.
In a previous publication,18 we described cell binding
experiments that evaluated the interaction of several tmFab-
SAv-Bi-MCP-111In complexes with SKOV-3 human ovarian
cancer cells that overexpress HER2. One of the MCPs in those
experiments was the Bi-PAAm (DTPA)50 polymer employed
here. In those experiments we compared radioactivity bound by
cells exposed to the Fab immunoconjugates to that for cells
coincubated with an excess of trastuzumab to determine the
relative proportion of total and nonspecific binding, and by
subtraction, the proportion of specific binding. Those experi-
Pendant Group Modification. In order to preserve
functionality at the polymer terminus during pendant group
modification and introduction of the DTPA metal chelating
groups, the dithiobenzoate group from the RAFT polymer-
ization was removed by aminolysis with excess ethanolamine in
THF (Scheme 2). The reaction was carried out in air to
promote oxidation of the thiol end groups to a disulfide, which
serves to protect the thiols during subsequent reactions of the
pendant groups. In the SEC chromatogram (Figure 1A), the
RAFT polymer after aminolysis and oxidation (dashed line)
shows a broader peak and an earlier elution time than the
precursor polymer (solid line). The dimeric disulfide-bridged
polymer was then treated with TFA in DCM to remove the
tBoc groups. The reaction was essentially quantitative. The
product polymer in the form of a polytrifluoroacetate salt was
recovered by precipitation in diethyl ether. Introduction of
DTPA groups followed a two-step protocol that we have
previously reported.18,25 In the first step, a large excess of
DTPA was activated with a limiting amount of DMTMM
(Scheme 2). Then a solution of the polymer in water was
added, keeping the pH at 8.5. Excess DTPA and other small
molecule byproducts were removed from the polymer by spin-
ments showed high specific binding (88.8
2.1%) and low
nonspecific binding for tmFab-SAv-DTPA-111In, but only about
50% specific binding for tmFab-SAv-MCP-111In. We concluded
that RICs with MCPs can target HER2 on the surface of
SKOV-3 cells but that nonspecific interactions occur.
In order to understand the binding affinity of the tmFab-SAv-
Bi-MCP complexes for HER2, we employed surface plasmon
resonance (SPR) measurements with the extracellular domain
(ECD) of HER2 covalently immobilized onto a gold sensor
chip. The association and dissociation rates were determined by
modeling the increase and decrease in SPR signals in the
sensorgrams (Figure 2). From the on-rates and off-rates, the
association (ka) and dissociation (kd) rate constants were
calculated. Association rate constants for tmFab-SAv-Bi-MCP
and tmFab-SAv-Bi-MCP-Dox were similar to that of tmFab-
SAv, and these values ranged from 1.2 × 105 to 1.9 × 105 M−1
s−1. The dissociation rate constants were also similar in
magnitude, ranging from 1.7 × 10−4 to 1.9 × 10−4 s−1. From the
ratio kd/ka = Kd, we determined that the dissociation constants
for HER2 binding were in the low nanomolar range (Table 1).
Based on the results of these experiments, we conclude that
neither the polymer nor the terminal Dox group had a
significant effect on the association rate, the dissociation rate, or
the dissociation constant for the binding of SAv-tmFab
1
filtration. The H NMR spectrum of this polymer is presented
in Figure 1B.
In the final step of the synthesis, the disulfides bridging the
dimeric MCPs were reduced with TCEP to thiols. An aliquot
was taken for analysis by SEC, and then Dox-Maleimide was
added in situ. We chose TCEP as the reducing agent because
the thiol addition to Dox-maleimide could be carried out
without first purifying the polymer to remove excess reducing
agent. The TCEP treated MCP showed a narrower peak and
longer retention time than the disulfide-bridged MCP (Figure
1C). An aliquot of the final heterotelechelic MCP was then
labeled with 111In and characterized by SEC-HPLC equipped
720
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
constants for intact antibodies with those of Fab fragments
because the intact antibody exhibits bivalent binding whereas
Fab fragments are monovalent, it is interesting to note that the
Fab complexes we examined have binding constants not
substantially different from trastuzumab IgG. Previously, it was
reported that the Kd values of trastuzumab and its Fab fragment
were similar for binding HER2 in cell binding assays.29 Thus,
we conclude that tmFab-SAv-Bi-MCP complexes have
sufficiently strong antigen recognition to deliver these RICs
to cancer cells that overexpress HER2.
Intracellular Localization of Immunoconjugates. Con-
focal Fluorescence Microscopy. Exploiting the intrinsic
fluorescence of Dox, we examined the cellular uptake and
intracellular distribution of tmFab-SAv-Bi-MCP-Dox by con-
focal fluorescence microscopy. Images are shown in Figure 3.
Figure 2. SPR plots of the binding and dissociation of tmFab-SAv
complexes to an SPR gold chip coated with the ECD of HER2: (A)
tmFab-SAv; (B) tmFab-SAv-Bi-MCP-Dox; (C) tmFab-SAv-Bi-MCP.
In all three groups, five different concentrations were employed for the
measurements, prepared by serially diluting the highest concentration
by 2-fold. The order of experiments was from lowest concentration to
highest. The highest concentration was 246 nM in (A) and 122 nM for
both (B) and (C).
Figure 3. Individual and merged confocal fluorescence microscopy
images of SK-BR-3 cells after incubating with tmFab-SAv-Bi-MCP-
Dox overnight and counterstaining: (A) The cell nuclei stained with
Hoechst 33342; (B) The cell membranes stained with WGA-Alexa
immunoconjugates; (C) Dox fluorescence; (D) The merged
fluorophore images.
Table 1. Binding Kinetics Data from SPR Experiments at 25
°C
a
b
10−5 × ka
104 × kd
c
d
analyte
(M−1 s−1
)
(s−1
)
KD (nM)
Rmax
The blue channel (Figure 3A) shows the cell nuclei
(counterstained with Hoechst 33342), the red channel (Figure
3B) shows the cell membranes [stained with wheat germ
agglutinin-Alexa Fluor 594 (WGA-Alexa)], and the green
channel (Figure 3C) shows the location of the Dox-containing
complexes. Figure 3D shows a superposition of all three
fluorophores. From this image, one can tell that Dox
fluorescence is localized inside the cell, close to the inner
surface of the cell membrane. Dox fluorescence was found
mainly in the cytoplasm and not in the cell nucleus. The Dox
containing complexes exhibited punctate fluorescence, which
may indicate endosome entrapment.30 This type of staining is
consistent with receptor-mediated endocytosis, which is likely
due to the tmFab-HER2 interaction.31
tmFab-SAv
1.17 0.01
1.49 0.01
1.79 0.03
1.92 0.06
1.53 0.03
1.29 0.04
109.96
82.24
tmFab-SAv-Bi-
MCP-Dox
tmFab-SAv-Bi-
MCP
1.88 0.01
1.65 0.04
0.88 0.03
90.67
a
b
Values of the second order rate constant for the on-rate. Values of
c
the first order dissociation rate constant. Values of the dissociation
equilibrium constant. Maximum response value.
d
immunoconjugates to the ECD of HER2. Although the
absolute values for parameters describing the binding of SAv-
tmFab immunoconjugates to the ECD of HER2 immobilized
on gold chips is not directly comparable to those for binding to
HER2 on cell membranes, the study within this SPR series
allows us to compare the relative binding of the different
complexes.26
Trastuzumab has been reported to bind to the ECD of HER2
with dissociation constant values Kd ranging from nanomolar to
subnanomolar.27,28 While it is difficult to compare binding
The observation of cell internalization of tmFab is surprising,
because it is well-known that endocytosis of Fab fragments is
less efficient than endocytosis of intact antibodies.31 This
difference is thought to be related to the promotion of HER2
receptor clustering due to bivalent binding of intact antibodies,
721
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
which is a requirement for the subsequent internalization. In
Figure 3, the observation of extensive cell internalization of the
tmFab-SAv-Bi-MCP-Dox complexes may be due to the
properties of the MCP which encourage cellular penetration.
In Figure 4 we compare confocal fluorescence microscopy
images of the tmFab-SAv-Bi-MCP-Dox-exposed cells with
uptake and nuclear localization, as discussed below. In Figure
4C we show results for cells treated with free Dox. Here there is
a higher Dox concentration in the cell nucleus, and Dox is also
found in the cytoplasm. The high relative intensity of free Dox
in the cell nucleus compared to the cytoplasm or cell
membrane is consistent with Dox interacting with proteasomes,
which enables its transport into the nucleus.20
Cell Fractionation Studies. Although the confocal fluo-
rescence images offer qualitative observations about tmFab-
SAv-Bi-MCP-Dox internalization into SK-BR-3 cells, more
quantitative information could be obtained through cell
fractionation experiments using the 111In-labeled complexes.
Three RICs were compared: (i) tmFab-SAv-Bi-DTPA without
MCPs, (ii) tmFab-SAv-Bi-MCP, and (iii) tmFab-SAv-Bi-MCP-
Dox. The results of these cell fractionation experiments are
summarized in Figure 5. All three RICs had the greatest
Figure 5. Percentage of RICs radioactivity localized in different cell
compartments of SK-BR-3 cells for 111In labeled tmFab-SAv-Bi-DTPA
(black column), tmFab-SAv-Bi-MCP-Dox (white column), and
tmFab-SAv-Bi-MCP (patterned column). The radioactivity in the
nucleus was fractionated into the radioactivity associated with the
chromatin and that associated with other components of the nucleus
(nuclear membrane, nucleoplasm). Values shown represent the mean
SEM, n = 3. Significant differences are p1 = 0.0108, p2 = 0.00166,
and p3 = 0.0104.
Figure 4. Images of SK-BR-3 cells incubated with tmFab-SAv-Bi-
MCP-Dox (A) without pretreatment with trastuzumab and (B) after
presaturation of HER2 with 100-fold excess of trastuzumab. Dox
fluorescence is shown in green, whereas the cell membrane is stained
red and the nucleus is stained blue (C) an image of the SK-BR-3 cells
incubated with free Dox showing more diffuse cytoplasmic staining as
well as nuclear uptake.
accumulation on the cell membrane. For example, 72.6 0.5%
of the total radioactivity for tmFab-SAv-Bi-DTPA was
associated with the cell membrane, higher than that of
images obtained from two control experiments. The concen-
tration of Dox in these experiments was monitored by UV−vis
absorption at 480 nm to ensure that the same amount of Dox
was used in each case. Figure 4A provides a second image
similar to that shown in Figure 3D for the combined
fluorescence of Dox as well as that of the membrane stain
and the nuclear stain. In Figure 4B we show an image of control
cells that were pretreated with an excess of trastuzumab to
block HER2 binding and then incubated with tmFab-SAv-Bi-
MCP-Dox. As in the case of Figure 4A, the Dox emission is
mainly localized in the cell cytoplasm, but the overall emission
signal from Dox is much weaker than in Figure 4A, reflecting
the decreased uptake of tmFab-SAv-Bi-MCP-Dox due to
blockage of the HER2 receptors, thus, demonstrating that the
binding and internalization of tmFab-SAv-Bi-MCP-Dox was
HER2-specific. The MCPs in these experiments were saturated
with stable In3+. At pH 7.4, each pendant group with its DTPA-
In chelate carries a net charge of −1. These charges along the
backbone of the MCP may have an important effect on cell
tmFab-SAv-Bi-MCP (52.8
SAv-Bi-MCP-Dox (65.2
significant differences in the amount of internalized radio-
activity. Of the tmFab-SAv-Bi-DTPA bound to the cells, 22
0.3% was present in the cytoplasm, with 5.5
nucleus, almost all of which (i.e., 5.0 0.3%) was associated
with the chromatin. tmFab-SAv-Bi-MCP-Dox had an almost
equal distribution in the cytoplasm (17.0 0.3%) and in the
0.5%, p = 0.0056) and tmFab-
0.7%, p = 0.0012). There were
0.3% in the
nucleus (17.8
1.0%) with 13.4
0.9% of the total
radioactivity in association with chromatin. The tmFab-SAv
complex with the MCP but without the terminal Dox (tmFab-
SAv-Bi-MCP) had the highest internalized fraction as well as
the highest amount of nuclear localization, with 32.0 0.9%
nuclear uptake (24.4
0.6% in the chromatin fraction) and
15.2 0.6% remaining in the cytosol. The values of the
distribution percentage for the different RICs are collected in
Table S1.
There are several points to highlight based on the data shown
in Figure 5. First, although all three RICs exhibit different
722
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
distribution profiles, they all have the greatest accumulation on
the cell surface. It is known that receptor-mediated internal-
ization is normally promoted by bivalent binding. In our case,
the use of Fab fragments in the RICs might compromise the
internalization ability.32 Second, the short penetration range of
Auger electrons accompanying radioactive decay of 111In means
that complexes carrying 111In will be most effective at killing
cancer cells when the decay occurs in proximity of the nuclear
DNA. Our finding that both MCP-containing complexes are
accumulated more efficiently in the cell nucleus and have a
strong tendency to associate with the chromatin is an intriguing
and promising finding for the application of these polymers to
deliver 111In to DNA for Auger electron radiotherapy of cancer.
Third, we were surprised to find that the terminal Dox group
on tmFab-SAv-Bi-MCP-Dox led to less nuclear localization
than the essentially identical polymer (tmFab-SAv-Bi-MCP)
without Dox. The protonated amine group on doxorubicin may
be necessary for the formation of Dox-proteasome complex and
subsequent transport of Dox into the nucleus.33,34 An
alternative polymer with the Dox attached as an alkylamine22
or through the ketone as a hydrazide might be a more effective
strategy for further promoting nuclear localization. From this
point of view, it may be the way we attached the terminal Dox
group to the polymer that interfered with its ability to transport
the complexes to the cell nucleus and the DNA.
experiments of Majonis et al.25 that at pH 7.4 (PBS) the mean
charge on each DTPA is between −2 to −3. These carboxylates
will be partially protonated at the more acidic pH of endosomes
and, especially, lysosomes. When the polymer is saturated with
trivalent metal ions, each repeat unit carries a charge of −1 (−4
for the 4 −COO(−) groups of DTPA, balanced by the +3
charge of the In3+ ion). Because of the tight binding, this metal
chelate is not as susceptible to protonation as the free
carboxylate ions of DTPA. Experiments with the In-saturated
polymer should indicate whether the charge along the polymer
backbone affects cell uptake and nuclear localization.
The results of these experiments are presented in Figure 6.
The tmFab-SAv-Bi-MCP complexes containing the indium-
Others have reported that trastuzumab, when conjugated to
charged macromolecules, undergoes enhanced uptake in SK-
BR-3 cells. Miyano et al.35 modified a G6 lysine dendrimer with
glutamate to generate an anionic structure. They subsequently
labeled this dendrimer with a fluorescent dye and conjugated it
to trastuzumab. These dendrimer-modified trastuzumab
showed more rapidly internalization to SK-BR-3 cells than
dye-labeled trastuzumab. Chan et al. conjugated trastuzumab
with a DTPA-derivatized G4 PAMAM dendrimer and labeled it
with 111In. In a cell fractionation study, this antibody−
dendrimer conjugate was compared with an identical conjugate
modified with an NLS-containing peptide. Both constructs
showed similar levels of nuclear uptake. The results showed
that the presence of dendrimer promoted cell uptake as well as
nuclear importation in SK-BR-3 cells and that conjugation of
the NLS peptide did not show any improvement.36
The fact that a significant fraction of the tmFab-SAv-Bi-MCP
complexes reach the nucleus points to a mechanism that
promotes endosomal release. It has been proposed that anionic
polymers are responsive to the acidic conditions inside the
endosome, and protonation of the polymer leads to
morphology changes that subsequently destabilize the endo-
somal membrane.37−39 For example, anionic polyelectrolytes
have been used to deliver antibody-polymer complexes,
oligonucleotides, and other biomolecules into the cell
cytoplasm by promoting escape from endosomes.40−42 It is
possible that the polyanionic nature of the MCPs similarly
destabilizes endosomes, promotes release of the tmFab-SAv-Bi-
MCP complexes into the cytoplasm and enhances the
opportunity for accumulation in the cell nucleus.
Figure 6. Percentage of radioactivity localized in different cell
compartments of SK-BR-3 cells for 111In labeled tmFab-SAv-Bi-MCP
with stable indium ion saturation of uncomplexed DTPA groups
(black column), tmFab-SAv- Bi-MCP without stable indium ion
saturation (white column). Error bars represent the mean SEM, n =
3. Significant differences are p1 = 0.0117, p2 = 0.0137, p3 = 0.00514.
saturated polymer have a higher probability of remaining at the
surface of SK-BR-3 cells (79.2 1.2%) than those with metal-
free DTPA pendant groups (68.7 0.8%; p = 0.0117). Of the
fraction taken up by the cells, the fraction of total radioactivity
found in the nucleus for the In-saturated polymer (10.3
0.5%) is only about half that of the complexes with metal-free
DTPA pendant groups (25.0 0.6%). We conclude that the
larger number of negative charges on the polymer not saturated
with In3+ ions leads to a significant enhancement of nuclear
localization compared to the In-saturated polymer complexes,
perhaps because of a higher probability of endosomal escape
once internalized.
It is interesting that the decrease of negative charges of the
polymers due to indium saturation promoted cell membrane
retention of tmFab-SAv-Bi-MCP complexes. This might relate
to the negative charges on the cell surface, which one
anticipates would have a larger repulsive interaction with the
MCPs without stable indium saturation than with the MCPs
with indium saturation. However, the lower nuclear uptake of
the indium saturated MCP-containing complexes suggests that
the negative charges elevate their nuclear uptake. This is
different from the nuclear uptake mechanism of NLS peptides,
which provide positive charges to interact with importins.43 In
summary, the effect of charged polymers in cell internalization
is still not well understood. We have much to learn about the
effect of polymer charge on interaction with cells and on
nuclear localization. The use of MCPs which can be
radiolabeled with 111In facilitates quantitative studies to further
understand these phenomena.
In order to evaluate the effects of charge on nuclear uptake,
we carried out an additional set of experiments with a sample of
tmFab-SAv-Bi-MCP that was saturated with stable In3+ after
being labeled with 111In. We have previously shown that there is
no loss of free 111In3+ ions under these conditions.14−16 The
MCP in tmFab-SAv-Bi-MCP carries approximately 50 DTPA
pendant groups. When labeled with 111In3+, only a small
fraction of these groups bind In ions. We can infer from the
723
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
SUMMARY AND CONCLUSIONS
ACKNOWLEDGMENTS
■
■
Through postpolymerization modification of polymers synthe-
sized by RAFT polymerization, we synthesized two well-
defined Bi-MCPs, both with a mean degree of polymerization
DPn ≈ 50 and with a biotin (Bi) at the initiating end. Metal
chelation was provided by DTPA pendant groups on every
monomer unit. One of the polymers (Bi-MCP-Dox) also
carried a doxorubicin at the chain terminus, attached through
its amino group as an amide. These polymers were conjugated
in a 1:1 molar ratio to streptavidin (tmFab-SAv) covalently
linked to a trastuzumab Fab fragment through a PEG24 spacer.
Radiolabeled complexes were formed by incubating tmFab-
SAv-Bi-MCP and tmFab-SAv-Bi-MCP-Dox with 111In. A
derivative of DTPA monofunctionalized with biotin (Bi-
DTPA) was synthesized for comparison with the MCP
complexes linked to tmFab-SAv and labeled with 111In.
SPR measurements for tmFab-SAv, tmFab-SAv-Bi-MCP-
Dox, and tmFab-SAv-Bi-MCP revealed that neither the
complexation of polymer nor the presence of the terminal
Dox significantly affected the binding affinity between the
tmFab and ECD of HER2.
Confocal microscopy measurements showed that tmFab-
SAv-Bi-MCP-Dox was taken up by SK-BR-3 cells, and a
significant fraction of these complexes entered the nucleus.
Pretreatment with excess trastuzumab reduced cellular uptake.
In parallel, we carried out cell fractionation studies on tmFab-
SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with
111In. Both radiolabeled complexes showed cell internalization
and nuclear localization. We conclude that metal-chelating
polymers with this composition appear to encourage internal-
ization, nuclear uptake, and chromatin (DNA) binding of
trastuzumab fragments modified with streptavidin in human
breast cancer cells expressing HER2. Covalent attachment of
doxorubicin to the MCPs actually led to a small reduction in
cell uptake and nuclear uptake. Further study is needed to
understand the impact of polymer charges on cellular uptake
and distribution to intracellular compartments.
The authors acknowledge the financial support from the
Canadian Institutes of Health Research/Natural Sciences and
Engineering Research Council Collaborative Health Research
Program (Grant No. CHRPJ 365423-09; CPG-95268) and
from DVS Sciences, Inc. We also thank Mr. Serge Zaretsky,
who assisted with the HPLC-MS measurements, and Mr. Nick
Jarvik for his assistance with the SPR experiments as well as
helpful discussions.
REFERENCES
■
(1) Shepard, H. M.; Lewis, G. D.; Sarup, J. C.; Fendly, B. M.;
Maneval, D.; Mordenti, J.; Figari, I.; Kotts, C. E.; Palladino, M. A., Jr.;
Ullrich, A. J. Clin. Immunol. 1991, 11, 117−127.
(2) Kapitanovic, S.; Radosevic, S.; Kapitanovic, M.; Andelinovic, S.;
Ferencic, Z.; Tavassoli, M.; Primorac, D.; Sonicki, Z.; Spaventi, S.;
Pavelic, K.; et al. Gastroenterology 1997, 112, 1103−1113.
(3) Maguire, H. C.; Greene, M. I. Semin. Oncol 1989, 16, 148−155.
(4) Press, M. F.; Pike, M. C.; Hung, G.; Zhou, J. Y.; Ma, Y.; George,
J.; Dietz-Band, J.; James, W.; Slamon, D. J.; Batsakis, J. G. Cancer Res.
1994, 54, 5675−5682.
(5) Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.;
McGuire, W. L. Science 1987, 235, 177−182.
(6) Di Fiore, P. P.; Pierce, J. H.; Kraus, M. H.; Segatto, O.; King, C.
R.; Aaronson, S. A. Science 1987, 237, 178−182.
(7) Hudziak, R. M.; Lewis, G. D.; Holmes, W. E.; Ullrich, A.;
Shepard, H. M. Cell Growth Differ. 1990, 1, 129−134.
(8) (a) Batra, J. K.; Kasprzyk, P. G.; Bird, R. E.; Pastan, I.; King, C. R.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5867−5871. (b) Rosenblum, M.
G.; Shawver, L. K.; Marks, J. W.; Brink, J.; Cheung, L.; Langton-
Webster, B. Clin. Cancer Res. 1999, 5, 865−874. (c) Wels, W.;
Harwerth, I.-M.; Mueller, M.; Groner, B.; Hynes, N. E. Cancer Res.
1992, 52, 6310−6317.
(9) Carter, P.; Presta, L.; Gorman, C. M.; Ridgway, J. B.; Henner, D.;
Wong, W. L.; Rowland, A. M.; Kotts, C. M. E. C.; Carver, M. E.;
Shepard, H. M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4285−4289.
(10) Nahta, R.; Esteva, F. J. Cancer Lett. 2006, 232, 123−138.
(11) Rosenblum, M. G.; Horn, S. A.; Cheung, L. H. Int. J. Cancer
2000, 88, 267−273.
(12) Coyne, C. P.; Ross, M. K.; Bailey, J. G. J. Drug Targeting 2009,
17, 474−489.
ASSOCIATED CONTENT
■
S
* Supporting Information
(13) Lambert, J. M. Curr. Opin. Pharmacol. 2005, 5, 543−549.
(14) Phillips, G. D. L.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons,
Additional experimental details, including instrumentation,
synthesis of Bi-EO2NH2, doxorubicin-maleimide, and of
monobiotin-DTPA and Bi-PAAm (DTPA)50-disulfide (polymer
4); 1H NMR spectra of Bi-PBocNAAm-50 (polymer 1) and Bi-
DTPA, HPLC-MS chromatograms of Bi-EO2NH2, p-SCN-Bn-
DTPA, Bi-DTPA before and after purification; doxorubicin and
Dox-maleimide; ESI and MALDI-TOF mass spectra of Dox-
maleimide; HPLC chromatograms of Bi-MCP-Dox, tmFab-
SAv, and tmFab-SAv-Bi-MCP-Dox; tables with results of cell
fractionation studies of tmFab-SAv-Bi-DTPA, tmFab-SAv-Bi-
MCP-Dox, and tmFab-SAv-Bi-MCP in SK-BR-3 cells. This
material is available free of charge via the Internet at http://
pubs.acs.org.
K. L.; Mai, E.; Blattler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.
̈
Cancer Res. 2008, 68, 9280−9290.
(15) Costantini, D. L.; Chan, C.; Cai, Z.; Vallis, K. A.; Reilly, R. M. J.
Nucl. Med. 2007, 48, 1357−1368.
(16) Torchilin, V. P. Bioconjugate Chem. 1998, 10, 146−149.
(17) Boyle, A. J.; Liu, P.; Lu, Y.; Weinrich, D.; Scollard, D. A.; Ngo
Njock Mbong, G.; Winnik, M. A.; Reilly, R. M. Pharm. Res. 2013, 30,
104−16.
(18) Liu, P.; Boyle, A. J.; Lu, Y.; Reilly, R. M.; Winnik, M. A.
Biomacromolecules 2012, 13, 2831−42.
(19) Lu, Y.; Chau, M.; Boyle, A. J.; Liu, P.; Niehoff, A.; Weinrich, D.;
Reilly, R. M.; Winnik, M. A. Biomacromolecules 2012, 13, 1296−1306.
(20) Kiyomiya, K.; Matsuo, S.; Kurebe, M. Cancer Res. 2001, 61,
2467−2471.
(21) Costantini, D. L.; Villani, D. F.; Vallis, K. A.; Reilly, R. M. J.
Nucl. Med. 2010, 51, 477−483.
(22) Ickenstein, L. M.; Edwards, K.; Sjoberg, S.; Carlsson, J.; Gedda,
AUTHOR INFORMATION
■
̈
Corresponding Author
*E-mail: raymond.reilly@utoronto.ca; mwinnik@chem.
utoronto.ca.
L. Nucl. Med. Biol. 2006, 33, 773−783.
(23) Colwill, K.; Graslund, S. Nat. Methods 2011, 8, 551−558.
̈
(24) Reilly, R. M.; Kiarash, R.; Cameron, R. G.; Porlier, N.; Sandhu,
Notes
J.; Hill, R. P.; Vallis, K.; Hendler, A.; Gariep
429−438.
́
y, J. J. Nucl. Med. 2000, 41,
The authors declare no competing financial interest.
724
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725

Biomacromolecules
Article
(25) Majonis, D.; Herrera, I.; Ornatsky, O.; Schulze, M.; Lou, X.;
Soleimani, M.; Nitz, M.; Winnik, M. A. Anal. Chem. 2010, 82, 8961−
8969.
(26) Vaneycken, I.; Devoogdt, N.; Van Gassen, N.; Vincke, C.;
Xavier, C.; Wernery, U.; Muyldermans, S.; Lahoutte, T.; Caveliers, V.
FASEB J. 2011, 25, 2433−2446.
(27) Troise, F.; Cafaro, V.; Giancola, C.; D’Alessio, G.; De Lorenzo,
C. FEBS J. 2008, 275, 4967−4979.
(28) Bostrom, J.; Haber, L.; Koenig, P.; Kelley, R. F.; Fuh, G. PloS
One 2011, 6, e17887.
(29) Chan, C.; Scollard, D. A.; McLarty, K.; Smith, S.; Reilly, R. M.
EJNMMI Res. 2011, 1, 15.
(30) (a) Bagalkot, V.; Farokhzad, O. C.; Langer, R.; Jon, S. Angew.
́
Chem., Int. Ed. 2006, 45, 8149−8152. (b) Gillies, E. R.; Frechet, J. M. J.
Bioconjugate Chem. 2005, 16, 361−368.
(31) Zhu, W.; Okollie, B.; Artemov, D. Cancer Biol. Ther. 2007, 6,
1960−1966.
(32) Fan, Z.; Lu, Y.; Wu, X.; Mendelsohn, J. J. Biol. Chem. 1994, 269,
27595−27602.
(33) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L.
Pharmacol. Rev. 2004, 56, 185−229.
(34) Kiyomiya, K.; Matsuo, S.; Kurebe, M. Life Sci. 1998, 62, 1853−
1860.
(35) Miyano, T.; Wijagkanalan, W.; Kawakami, S.; Yamashita, F.;
Hashida, M. Mol. Pharmaceutics 2010, 7, 1318−1327.
(36) Chan, C.; Cai, Z.; Reilly, R. Pharm. Res. 2013, 30, 1999−2009.
(37) Yessine, M.-A.; Leroux, J.-C. Adv. Drug Delivery Rev. 2004, 56,
999−1021.
(38) Breunig, M.; Bauer, S.; Goepferich, A. Eur. J. Pharm. Biopharm.
2008, 68, 112−128.
(39) Lackey, C. A.; Murthy, N.; Press, O. W.; Tirrell, D. A.; Hoffman,
A. S.; Stayton, P. S. Bioconjugate Chem. 1999, 10, 401−405.
(40) Bulmus, V.; Woodward, M.; Lin, L.; Murthy, N.; Stayton, P.;
Hoffman, A. J. Controlled Release 2003, 93, 105−120.
(41) Lackey, C. A.; Press, O. W.; Hoffman, A. S.; Stayton, P. S.
Bioconjugate Chem. 2002, 13, 996−1001.
(42) Berguig, G. Y.; Convertine, A. J.; Shi, J.; Palanca-Wessels, M. C.;
Duvall, C. L.; Pun, S. H.; Press, O. W.; Stayton, P. S. Mol. Pharm.
2012, 9, 3506−3514.
(43) Costantini, D. L.; Hu, M.; Reilly, R. M. Cancer Biother.
Radiopharm. 2008, 23, 3−24.
725
dx.doi.org/10.1021/bm401483a | Biomacromolecules 2014, 15, 715−725