Targeted theranostic platinum(IV) prodrug with a built-in aggregation-induced emission light-up apoptosis sensor for noninvasive early evaluation of its therapeutic responses in situ

Article abstract of DOI:10.1021/ja411811w

Targeted drug delivery to tumor cells with minimized side effects and real-time in situ monitoring of drug efficacy is highly desirable for personalized medicine. In this work, we report the synthesis and biological evaluation of a chemotherapeutic Pt(IV)

Full text of DOI:10.1021/ja411811w

Article
pubs.acs.org/JACS
Targeted Theranostic Platinum(IV) Prodrug with a Built-In
Aggregation-Induced Emission Light-Up Apoptosis Sensor for
Noninvasive Early Evaluation of Its Therapeutic Responses in Situ
Youyong Yuan,† Ryan T. K. Kwok,‡ Ben Zhong Tang,*,‡,§ and Bin Liu*,†,∥
†Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585
‡Department of Chemistry, Institute of Molecular Functional Materials, Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
§SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials
and Devices, South China University of Technology, Guangzhou China 510640
∥Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602
S
* Supporting Information
ABSTRACT: Targeted drug delivery to tumor cells with
minimized side effects and real-time in situ monitoring of drug
efficacy is highly desirable for personalized medicine. In this
work, we report the synthesis and biological evaluation of a
chemotherapeutic Pt(IV) prodrug whose two axial positions
are functionalized with a cyclic arginine−glycine−aspartic acid
(cRGD) tripeptide for targeting integrin αvβ3 overexpressed
cancer cells and an apoptosis sensor which is composed of
tetraphenylsilole (TPS) fluorophore with aggregation-induced
emission (AIE) characteristics and a caspase-3 enzyme specific
Asp-Glu-Val-Asp (DEVD) peptide. The targeted Pt(IV) prodrug can selectively bind to αvβ3 integrin overexpressed cancer cells
to facilitate cellular uptake. In addition, the Pt(IV) prodrug can be reduced to active Pt(II) drug in cells and release the apoptosis
sensor TPS-DEVD simultaneously. The reduced Pt(II) drug can induce the cell apoptosis and activate caspase-3 enzyme to
cleave the DEVD peptide sequence. Due to free rotation of the phenylene rings, TPS-DEVD is nonemissive in aqueous media.
The specific cleavage of DEVD by caspase-3 generates the hydrophobic TPS residue, which tends to aggregate, resulting in
restriction of intramolecular rotations of the phenyl rings and ultimately leading to fluorescence enhancement. Such noninvasive
and real-time imaging of drug-induced apoptosis in situ can be used as an indicator for early evaluation of the therapeutic
responses of a specific anticancer drug.
no direct information is given for the therapeutic effect, which
requires further experiments to evaluate. The early evaluation of
cancer’s response to a specific therapy is of high importance in
clinical applications because it can minimize the duration of
ineffective courses in cancer therapy. Currently, the common
method to evaluate the effectiveness of cancer treatment is by
measuring the tumor size using magnetic resonance imaging
(MRI).4 However, MRI technique is unsatisfactory in
evaluating the early response of cancer to a particular
therapeutic drug as the change in tumor size is not obvious
at the early stages of therapy. As a consequence, it is highly
desirable if one could design and develop a theranostic system
that can simultaneously deliver the therapeutic drugs and
noninvasively evaluate the therapeutic responses in situ.
INTRODUCTION
■
Drug delivery systems have been widely used for improving
cancer therapeutic efficiencies of small-molecule drugs.1
Typically, the anticancer drugs are loaded or covalently
conjugated to the delivery systems and are expected to release
only at specific tumor sites. Development of strategies for real-
time monitoring of drug delivery/release at target cancer cells is
a very important step to indirectly evaluate the potential
therapeutic efficiency.2 The most common strategy is to use
fluorescent dyes/drugs as model cargo systems. However, such
strategy faces some problems, e.g. difficulties in correlating the
release of the fluorescent dyes to the actual drug molecules, or
the restricted usage of limited fluorescent drugs (e.g.,
doxorubicin). Recently, some theranostic prodrug delivery
systems have been developed for real-time monitoring of the
release of active drugs by conjugating the drugs with fluorescent
dyes.3 The design strategy relies on drug release-induced
fluorescence intensity or color change upon tumor-associated
stimulus. Most of the systems reported so far are focused on
monitoring the delivery and/or release of the drugs. However,
One of the most promising solutions to this issue is to
incorporate an apoptosis sensor into the system. As therapeutic
drugs generally kill the cancer cells by activating apoptosis
Received: November 20, 2013
Published: January 17, 2014
© 2014 American Chemical Society
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Scheme 1. Schematic Illustration of the Targeted Theranostic Platinum(IV) Prodrug with a Built-In Aggregation-Induced
Emission (AIE) Light-Up Apoptosis Sensor for Noninvasive in Situ Early Evaluation of Its Therapeutic Responses
within a short period of time, the apoptosis sensor could
evaluate the therapy efficacy at a very early stage.5 So far, several
strategies have been utilized for detection of cell apoptosis on
the basis of the alterations of apoptosis-associated biochemical
substances within the cells.6 One of the most important
pathways of therapeutic drug-induced apoptosis is activation of
caspase-3, a cysteine protease which specifically cleaves proteins
containing the Asp-Glu-Val-Asp (DEVD) peptide sequence.7
Recently, we have developed a simple strategy for real-time
monitoring of cell apoptosis in vitro and in vivo based on
probes containing fluorogens with aggregation-induced emis-
sion (AIE) characteristics.8 These probes are nonemissive in
molecularly dissolved stated, but the caspase-3 cleaved AIE
residues are highly emissive due to the restriction of
intramolecular rotations (RIR), which reduces energy dis-
sipation via nonradiative channels.9 Although these probes are
able to respond to drug-induced apoptosis, they are not able to
report the exact drug location or quantify the therapeutic
responses in situ as the probes and drugs may not be in the
same cells or the probe is not colocalized with the drug when
they are in the same cells. It is highly desirable to develop a
theranostic drug delivery system that could also evaluate the
therapeutic response in situ.
in the tumor site. The prodrug can also avoid undesirable
interactions with proteins and other biomolecules in blood
circulation to reduce the side effects. As a consequence,
targeted delivery of a Pt(IV) prodrug with a built-in apoptosis
sensor would be an ideal strategy for simultaneous drug
delivery with reduced side effects and noninvasive early
evaluation of its therapeutic responses in situ.
In this contribution, we developed a targetable theranostic
Pt(IV) prodrug with a special focus on monitoring drug-
induced cell apoptosis in situ. The system is composed of a
chemotherapeutic Pt(IV) prodrug which can be reduced to
active Pt(II) intracellularly, an apoptosis sensor (TPS-DEVD)
based on tetraphenylsilole (TPS) with AIE characteristics, and a
cyclic(RGD) peptide as the targeting ligand (Scheme 1). The
prodrug can accumulate preferentially in cancer cells with
overexpressed αvβ3 integrin and release the active drug Pt(II)
and apoptosis sensor TPS-DEVD upon the intracellular
reduction of the Pt(IV) prodrug. The released Pt(II) can
induce cell apoptosis and activate caspase-3 to cleave the
DEVD peptide in TPS-DEVD and trigger the fluorescence. The
fluorescence turn-on response allows us to utilize the
theranostic platform for real-time and noninvasive imaging of
the therapeutic responses of a specific anticancer drug.
Platinum drug is one of the most effective therapeutic agents
against many cancers.10 In clinics, cisplatin (cis-
dichlorodiammineplatinum(II)) is one of the most widely
used anticancer drugs which can bind to DNA and induce cell
apoptosis.11 However, like most of the other small-molecule
anticancer drugs, its clinical use is limited due to the severe side
effects.10b A promising strategy to minimize the side effect of
platinum drug is to employ targetable nontoxic Pt(IV)
complexes as prodrugs which can be intracellularly activated
by reduction to the toxic Pt(II) form.12 Receptor-targeted
chemotherapeutics are attractive because they target receptors
that are overexpressed in tumors so that they can minimize
systematic distribution of the drugs and facilitate their binding
and internalization in cells that express the specific receptor.1a
The greater inertness of Pt(IV) complexes makes the prodrug
more stable in blood and thus has the potential to accumulate
EXPERIMENTAL SECTION
■
General Information. Cisplatin, N,N-diisopropylethyl-
amine (DIEA), N-hydroxysuccinimide (NHS), 1-ethyl-3-[3-
dimethylaminopropyl]carbodiimide hydrochloride (EDC),
copper(II) sulfate (CuSO4), sodium ascorbate, ascorbic acid,
succinic anhydride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT), anhydrous dimethyl sulfoxide
(DMSO), anhydrous dimethylformamide (DMF), lithium
wires, naphthalene, 4-bromobenzene, 4-bromobenzyl bromide,
sodium azide, dichlorobis(triphenylphosphine)palladium(II),
ZnCl2·TMEDA, piperazine-N,N′-bis(2-ethanesulfonic acid)
(PIPES), diethyldithiocarbamate (DDTC), bovine serum
albumin (BSA), lysozyme, pepsin, trypsin and other chemicals
were all purchased from Sigma-Aldrich and used as received
without further purification. Hexane and tetrahydrofuran
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(THF) purchased from Fisher Scientific were distilled from
sodium benzophenoneketyl immediately prior to use. Dichloro-
methane (DCM) was distilled over calcium hydride.
Deuterated solvents with tetramethylsilane (TMS) as internal
reference were purchased from Cambridge Isotope Laborato-
ries Inc. Alkyne-functionalized DEVD (Asp-Glu-Val-Asp-Pra)
and amine-functionalized cRGD (cyclic(Arg-Gly-Asp-D-Phe-
Lys)) were customized from GL Biochem Ltd. cis,cis,trans-
Diamminedichlorodisuccinatoplatinum(IV) was synthesized
following a literature method.13
Dulbecco’s Modified Essential Medium (DMEM) is a
commercial product of National University Medical Institutes
(Singapore). Milli-Q water was supplied by Milli-Q Plus System
(Millipore Corporation, Breford, United States). Piperazine-
N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer containing 50
mM PIPES, 100 mM NaCl, 1 mM ethylenediaminetetraacetic
acid (EDTA), 0.1% w/v 3-[(3-cholamidopropyl)-
dimethylammonio] propanesulfonic and 25% w/v sucrose
(pH = 7.2). Recombinant human caspase-3 was purchased
from R&D Systems. Caspase-3 inhibitor 5-[(S)-(+)-2-
(methoxymethyl)pyrrolidino]sulfonylisatin was purchased
from Calbiochem. Fetal bovine serum (FBS) and trypsin−
EDTA solution were purchased from Gibco (Lige Technolo-
gies, AG, Switzerland). Staurosporine (STS) was purchased
from Biovision. DRAQ5 was purchased from Biostatus. Cleaved
caspase-3 (Asp175) (5A1E) rabbit mAb (#9664) was
purchased from Cell Signaling. Mouse anti-rabbit IgG-TR
(sc-3917) was purchased from Santa Cruz.
Characterization. NMR spectra were measured on a
Bruker ARX 400 NMR spectrometer. Chemical shifts were
reported in parts per million (ppm) referenced with respect to
residual solvent (CDCl3 = 7.26 ppm, (CD3)2SO = 2.50 ppm or
tetramethylsilane Si(CH3)4 = 0 ppm). Particle size and size
distribution were determined by laser light scattering (LLS)
with a particle size analyzer (90 Plus, Brookhaven Instruments
Co., United States) at a fixed angle of 90° at room temperature.
HPLC profiles and mass spectra were acquired using a
Shimadzu IT-TOF. A 0.1% TFA/H2O and 0.1% TFA/
acetonitrile were used as eluents for the HPLC experiments.
High-resolution mass spectra (HRMS) were recorded on a
Finnigan MAT TSQ 7000 mass spectrometer. UV−vis
absorption spectra were taken on a Milton Ray Spectronic
3000 array spectrophotometer. Photoluminescence (PL)
spectra were measured on a Perkin-Elmer LS 55 spectro-
fluorometer.
green solution of LiNaph. A solution of dimethylbis-
(phenylethynyl)silane (0.52 g, 2 mmol) in 5 mL of THF was
then added dropwise to LiNaph solution at room temperature.
After stirring for 1 h, the mixture was cooled to 0 °C and then
diluted with 25 mL of THF. A black suspension was formed
upon addition of ZnCl2·TMEDA (2 g, 8 mmol). After stirring
for an additional hour at room temperature, a solution
containing 4-bromobenzene (0.34 g, 2.2 mmol), 4-bromoben-
zyl azide (0.47 g, 2.2 mmol) and PdCl2(PPh3)2 (0.08 g, 0.1
mmol) in 25 mL of THF was added. The mixture was refluxed
overnight. After cooling down to room temperature, 100 mL of
1 M HCl solution was added and the mixture was extracted
with DCM several times. The organic layer was combined and
washed with brine and water and then dried over magnesium
sulfate. After solvent evaporation under reduced pressure, the
residue was purified by a silica-gel column using hexane as
eluent. The product was obtained as a yellow solid in 36% yield
(0.34 g). 1H NMR (CDCl3, 400 MHz), δ (TMS, ppm): 7.15−
7.06 (m, 6H, −CH−), 7.02−6.99 (m, 5H, −CH−), 6.95−6.93
(m, 4H, −CH−), 6.82−6.79 (m, 4H, −CH−), 4.23 (s, 2H,
−CH2−), 0.48 (s, 6H, −CH3). 13C NMR (CDCl3, 100 MHz),
δ (TMS, ppm): 154.5, 153.9, 142.1, 141.2, 140.1, 139.8, 138.7,
132.5, 130.0, 129.2, 128.9, 128.0, 127.5, 126.4, 126.3, 125.7,
54.7, −3.80. HRMS (MALDI-TOF): m/z 469.1959 (M+, calcd
469.1974).
Synthesis of N-Hydroxysuccinimide-Activated
Platinum(IV) Complexes. A mixture of platinum(IV)
complex cis, cis, trans-diamminedichlorodisuccinatoplatinum-
(IV) (32.1 mg, 0.06 mmol), EDC (23.0 mg, 0.12 mmol) and
NHS (13.8 mg, 0.12 mmol) in anhydrous DMF (1 mL) was
stirred at room temperature overnight. After that, the mixture
was purified by HPLC (solvent A: water with 0.1% TFA,
solvent B: CH3CN with 0.1% TFA) and quickly lyophilized to
yield the desired product as a white powder in 78% yield (34.1
1
mg). H NMR (400 MHz, DMF-d7), δ (TMS, ppm): 6.92−
6.68 (m, 6H, −NH3), 2.94−2.91 (m, 8H, −CH2−), 2.89−2.84
(m, 4H, −CH2−), 2.72−2.68 (m, 4H, −CH2−). 13C NMR
(DMF-d7, 100 MHz), δ (TMS, ppm): 178.5, 170.6, 168.8, 30.0,
27.1, 25.9. IT-TOF-MS: m/z [M + H]+ calcd 728.026, found
728.021.
“Click” Synthesis of Amine-Terminated TPS-DEVD.
Alkyne-functionalized DEVD (10.2 mg, 20 μmol) and TPS-
CH2N3 (9.4 mg, 20 μmol) were dissolved in a mixture of
DMSO/H2O solution (v/v = 1/1; 1.0 mL). The “click”
reaction was initiated by sequential addition of catalytic
amounts of CuSO4 (9.6 mg, 6 μmol) and sodium ascorbate
(2.4 mg, 12 μmol). The reaction was continued with shaking at
room temperature for another 24 h. The final product was
purified by HPLC and lyophilized under vacuum to yield the
Synthesis of 4-Bromobenzyl Azide. Into a flask equipped
with a magnetic stirrer were added 4-bromobenzyl bromide
(7.5 g, 30 mmol), sodium azide (7.8 g, 120 mmol), and 40 mL
of DMSO. After stirring at 70 °C for 12 h, the solution was
poured into 150 mL of water and extracted with DCM. The
crude product was purified by silica-gel chromatography using
hexane as eluent to give a colorless, viscous liquid in 96% yield
1
probe as white powders in 45% yield (9.4 mg). H NMR
(DMSO−d6, 400 MHz): 12.24 (s, 3H, −COOH), 8.49 (d, 1H,
−NH−), 8.32 (d, 1H, −NH−), 8.05 (d, 1H, −NH−), 7.92 (d,
1H, −NH−), 7.86 (s, 1H, −CH−), 7.22−7.06 (m, 6H,
−CH−), 7.02−6.99 (m, 5H, −CH−), 6.95−6.88 (m, 4H,
−CH−), 6.82−6.79 (m, 4H, −CH−), 5.45 (s, 2H, −CH2−),
4.54−4.49 (m, 1H, −CH−), 4.38 (m, 2H, −CH−), 4.17−4.13
(m, 2H, −CH−), 3.10−3.05 (m, 1H, −CH2−), 2.92−2.88 (m,
1H, −CH2−), 2.71−2.65 (m, 2H, −CH2−), 2.26−2.21 (m, 2H,
−CH2−), 2.01−1.85 (m, 2H, −CH2−), 0.84−0.74 (m, 6H,
−CH3), 0.43 (s, 6H, −CH3); IT-TOF-MS: m/z [M + H]+
calcd 1040.426, found 1040.866.
1
(6.12 g). H NMR (CDCl3, 400 MHz), δ (TMS, ppm): 7.47
(d, 2H, −CH−), 7.15 (d, 2H, −CH−), 4.26 (s, 2H, −CH2−).
13C NMR (CDCl3, 100 MHz), δ (TMS, ppm): 134.3, 131.8,
129.6, 122.1, 53.9. HRMS (MALDI-TOF): m/z 210.964 (M+,
calcd 210.974).
Synthesis of 1,1-Dimethyl-2-[4-(azidomethyl)phenyl]-
3,4,5-triphenylsilole (TPS-CH2N3). Dimethylbis-
(phenylethynyl)silane was prepared according to our published
procedures.14 A mixture of lithium (0.056 g, 8 mmol) and
naphthalene (1.04 g, 8 mmol) in 8 mL of THF was stirred at
room temperature under nitrogen for 3 h to form a deep, dark-
Synthesis of Theranostic Prodrug TPS-DEVD-Pt-cRGD.
Amine-terminated TPS-DEVD (9.0 mg, 8.7 μmol) and amine-
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functionalized cRGD (5.2 mg, 8.7 μmol) were dissolved in
anhydrous DMSO (1.0 mL) with a catalytic amount of DIEA
(1.0 μL). The mixture was stirred at room temperature for 10
min. Then N-hydroxysuccinimide-activated platinum(IV) com-
plex (6.3 mg, 8.7 μmol) in DMSO (0.5 mL) was added quickly
to the above mixture. The reaction was continued with stirring
at room temperature for another 24 h. The final product was
purified by HPLC and lyophilized under vacuum to yield the
buffer, and then incubated with mouse anti-rabbit IgG-TR (0.8
μg mL−1) in PBS for 1 h, followed by washing with PBS. The
cells were then imaged immediately by confocal laser scanning
microscope (CLSM, Zeiss LSM 410, Jena, Germany). The
images were analyzed by Image J 1.43 × program (developed
by NIH, http://rsbweb.nih.gov/ij/).
Quantification of Cell Apoptosis by Fluorescence
Microplate Reader. U87-MG and MCF-7 cells were seeded
in 96-well plates (Costar, United States) at an intensity of 4 ×
104 cells mL−1. After confluence, the medium was replaced by
different concentrations of TPS-DEVD-Pt-cRGD in fresh FBS-
free DMEM medium for 2 h. After the determined incubation
time at 37 °C, the adherent cells were washed twice with 1×
PBS buffer followed by fluorescence measurement using a T-
CAN microplate reader. The excitation and emission wave-
lengths are 365 and 480 nm, respectively.
Cytotoxicity of the Prodrug. 3-(4,5-Dimethythiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assays were used to
assess the metabolic activity of U87-MG and MCF-7 cancer
cells. The cells were seeded in 96-well plates (Costar, IL,
United States) at an intensity of 4 × 104 cells mL−1. After 24 h
incubation, the medium was replaced by the probe suspension
at different concentration of the prodrug and incubated at 37
°C for 6 h. After the designated time intervals, the wells were
washed twice with 1× PBS buffer, and 100 μL of freshly
prepared MTT (0.5 mg mL−1) solution in culture medium was
added into each well. The MTT medium solution was carefully
removed after 3 h incubation in the incubator at 37 °C. DMSO
(100 μL) was then added into each well and the plate was
gently shaken to dissolve all the precipitates formed. The
absorbance of MTT at 570 nm was monitored by the
microplate reader (Genios Tecan). Cell viability was expressed
by the ratio of absorbance of the cells incubated with prodrug
suspension to that of the cells incubated with culture medium
only.
1
prodrug as white powders in 40% yield (7.4 mg). H NMR
(DMSO-d6, 400 MHz): 12.24 (s, 3H, −COOH), 8.55 (d, 1H,
−NH−), 8.51 (d, 1H, −NH−), 8.31 (d, 1H, −NH−), 8.15−
8.05 (m, 6H, −NH−), 7.91 (d, 1H, −NH−), 7.86 (s, 1H,
−CH−), 7.62 (d, 2H, −NH−), 7.56 (d, 1H, −NH−), 7.45 (m,
1H, −NH−), 7.22−7.06 (m, 6H, −CH−), 7.02−6.99 (m, 5H,
−CH−), 6.95−6.88 (m, 4H, −CH−), 6.82−6.79 (m, 4H,
−CH−), 6.60−6.35 (m, 6H, −NH3), 5.45 (s, 2H, −CH2−),
4.65−4.60 (m, 1H, −CH−), 4.54−4.48 (m, 1H, −CH−),
4.40−4.32 (m, 3H, −CH−), 4.17−4.10 (m, 3H, −CH−),
4.05−4.02 (m, 1H, −CH−), 3.95−3.91 (m, 1H, −CH−),
3.10−3.06 (m, 4H, −CH2−), 2.95−2.87 (m, 2H, −CH2−),
2.85−2.78 (m, 2H, −CH2−), 2.75−2.62 (m, 6H, −CH2−),
2.50−2.45 (m, 8H, −CH2−), 2.27−2.23 (m, 4H, −CH2−),
1.93−1.89 (m, 4H, −CH2−), 1.72 (m, 2H, −CH2−), 1.41−
1.38 (m, 2H, −CH2−), 1.37−1.32 (m, 2H, −CH2−), 0.84−
0.74 (m, 6H, −CH3), 0.43 (s, 6H, −CH3); ESI-MS: m/z [M +
H]+ calcd 2141.719, found 2141.689.
General Procedure for Enzymatic Assay. DMSO stock
solutions of TPS-DEVD-Pt-cRGD were diluted with a mixture
of DMSO and PIPES (v/v = 1/199) to 10 μM. Next, each
probe was incubated with ascorbic acid or caspase-3 at room
temperature, and the change of fluorescence intensity was
measured. The PL spectra were collected from 420 to 650 nm
under excitation at 365 nm.
Cell Culture. U87-MG human glioblastoma cancer cells,
MCF-7 breast cancer cells, and 293T normal cells were
provided by American Type Culture Collection (ATCC). The
cells were cultured in DMEM (Invitrogen, Carlsbad, CA)
containing 10% heat-inactivated FBS (Invitrogen), 100 U/mL
penicillin, and 100 μg/mL streptomycin (Thermo Scientific)
and were maintained in a humidified incubator at 37 °C with
5% CO2. Before experiment, the cells were precultured until
confluence was reached.
Confocal Imaging. U87-MG, MCF-7, and 293T cells were
cultured in the chambers (LAB-TEK, Chambered Coverglass
System) at 37 °C. After 80% confluence, the culture medium
was removed, and the cells wefre washed twice with PBS buffer.
The probe in DMSO stock solution was then added to the
chamber to reach a final concentration of 5 μM. In some
experiments, the cells were preincubated with media containing
cRGD (50 μM) or inhibitor (5 μM) prior to prodrug
incubation. After incubation with the prodrug at 37 °C for 2
h, the medium was replaced with fresh medium and further
incubated for different times. Then the cells were washed twice
with ice-cold PBS, and the cell nuclei were live stained with
DRAQ5 (Biostatus) following the standard protocol of the
manufacturer. For colocalization with active caspase-3 antibody,
the cells were first fixed for 15 min with 3.7% formaldehyde in 1
× PBS at room temperature, washed twice with cold PBS again,
and permeabilized with 0.1% Triton X-100 in PBS for 10 min.
The cells were then blocked with 2% BSA in 1 × PBS for 30
min and washed twice with PBS. The cells were subsequently
incubated with a mixture of anti-caspase-3 antibody/PBS (v/v
= 1/99) for 1 h at room temperature, washed once with PBS
RESULTS AND DISCUSSION
■
Azide-functionalized tetraphenylsilole (TPS-CH2N3) was syn-
thesized by the heterobifunctional modification of dimethylbis-
(phenylethynyl)silane with 4-bromobenzene and 4-bromoben-
zyl azide. Detailed synthesis and characterization of TPS-
CH2N3 and the intermediates are shown in the Experimental
Section and Supporting Information (SI) (Scheme S1, Figures
S1−S2). The coupling between TPS-CH2N3 and alkyne-
functionalized DEVD via “click” reaction using CuSO4/sodium
ascorbate as the catalyst in DMSO/water (v/v = 1/1) afforded
amine terminated TPS-DEVD in 45% yield after HPLC
purification (Scheme S2, SI). The purity and identity of the
probe was well characterized by analytical HPLC, NMR, and
HRMS (Figures S3−S5, SI). Commercially available anticancer
drug cisplatin was modified to be used as the linker between
amine terminated TPS-DEVD and the amine functionalized
cRGD (Scheme S3, SI). In the first step, cisplatin was oxidized
by hydrogen peroxide to produce cis, cis, trans-
diaminedichlorodihydroxyplatinum(IV) complex. Next, the
Pt(IV) complex was reacted with succinic anhydride in
DMSO at 70 °C for 12 h to yield cis, cis, trans-
diamminedichlorodisuccinatoplatinum(IV) complex (Figures
S6−S7, SI). The activated Pt(IV) complex was subsequently
obtained by reacting the carboxylic acid groups with NHS in
anhydrous DMF using EDC as the coupling reagent. The
activated Pt(IV) linker was purified by HPLC and lyophilized
as white powder with 78% yield (Figures S8−S11, SI).
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Scheme 2. Synthetic Route to the Theranostic Prodrug of TPS-DEVD-Pt-cRGD
Asymmetric functionalization of activated Pt(IV) linker with
amine-terminated TPS-DEVD and amine-functionalized cRGD
in the presence of N,N-diisopropylethylamine (DIEA) in
anhydrous DMSO afforded the desired product, TPS-DEVD-
Pt-cRGD in 40% yield after HPLC purification (Scheme 2).
The purity and identity of the probe was well characterized by
HPLC, NMR, and HRMS (Figures S12−S14, SI).
For any prodrug, it is essential that it can be easily
transformed into its original form to restore its therapeutic
ability after the modification. To evaluate our prodrug as a
potential drug delivery system, we studied the nature of the
formed Pt(II) species upon reduction of the synthesized
prodrug. It is reported that diethyldithiocarbamate (DDTC) is
inert to Pt(IV) complexes, but can react with Pt(II) complexes
to yield the adduct of Pt(DDTC)2.15 In this work, we used
HPLC−MS to monitor the adduct formation for Pt(IV)
complexes before and after treatment with ascorbic acid in the
presence of DDTC. We chose ascorbic acid as a reduction
agent because its abundance in cells (1 mM), which has been
demonstrated to be a major substance for the reduction of
Pt(IV).12c As shown in Figure 1, cisplatin can efficiently bind to
DDTC to form the adduct of Pt(DDTC)2. The complex was
further confirmed by IT-TOF with a mass-to-charge ratio (m/
z) of 492.104 (Figure S15, SI). On the other hand, Pt(DDTC)2
is only formed when the prodrug is treated with ascorbic acid
and in the presence of DDTC, confirming that the released Pt
entities are indeed Pt(II) species. In addition, an apoptosis
sensor TPS-DEVD with an m/z of 1140.344 is formed after the
reduction (Figure S16, SI) and this fraction was collected and
lyophilized for further use. Based on these results, we confirm
that the Pt(IV) prodrug can be reduced in the presence of
ascorbic acid to generate the reactive Pt(II) drug and apoptosis
sensor simultaneously.
We next studied the optical properties of the prodrug. The
UV−vis absorption spectra of TPS-CH2N3 in THF and TPS-
DEVD-Pt-cRGD in DMSO/PIPES (v/v = 1/199) buffer are
shown in Figure S17A, SI. Both have a similar absorption
profile in the 320−440 nm range. It is known that AIE
fluorogen is nonemissive in good solvents but emits intensely in
solid or as aggregates in poor solvents.9b,c As can be seen from
the photoluminescence (PL) spectra shown in Figure 2A, TPS-
CH2N3 shows intense fluorescence in DMSO/PIPES (v/v = 1/
199), while the TPS-DEVD and TPS-DEVD-Pt-cRGD are
almost nonfluorescent in the same medium, due to their good
solubility in water. The aggregate formation for hydrophobic
TPS-CH2N3 in DMSO/PIPES (v/v = 1/199) buffer was
confirmed by laser light scattering (LLS) measurement, which
shows an average diameter of 118 nm (Figure S17B, SI). As
biosensing is often conducted in buffers, it is important to study
the effect of ionic strength on the emission behavior of the
prodrug. The experiments were performed by adding sodium
chloride into an aqueous solution of TPS-DEVD-Pt-cRGD or
TPS-DEVD (10 μM). Almost no change in fluorescence
intensity is observed for both when the concentration of NaCl
is increased from 0 to 960 mM (Figure S18A, SI). Their PL
profiles also do not change in the commonly used medium
Dulbecco’s Modified Eagle Medium (DMEM). Due to the low
pKa value of carboxyl groups in DEVD, the prodrug remains
almost nonfluorescent in slightly acidic conditions (pH 5.5,
Figure S18B, SI). Based on the above results, we know that
TPS-DEVD-Pt-cRGD and the apoptosis sensor TPS-DEVD
maintain an “off” state in the complex environment and thus
have great potential to serve as a specific light-up apoptosis
sensor for drug effect study with minimum background
interference.
Figure 1. HPLC spectra showing the reaction of diethyldithiocarba-
mate (DDTC) with cisplatin and reduced Pt(IV) prodrug: (A) DDTC
alone; (B) the product for reaction between DDTC and cisplatin; (C)
the mixture of DDTC and TPS-DEVD-Pt-cRGD; (D) the products
for the reaction between DDTC with TPS-DEVD-Pt-cRGD (10 μM)
after treated with 1 mM ascorbic acid for 12 h.
Figure 2B shows the optical properties of the prodrug before
and after reduction with ascorbic acid. There is no fluorescence
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Figure 2. (A) PL spectra of TPS-CH2N3, apoptosis sensor TPS-DEVD, TPS-DEVD-Pt-cRGD in DMSO/PIPES (v/v = 1/199). Inset:
Corresponding photographs taken under illumination of a UV lamp at 365 nm. (B) PL spectra of TPS-DEVD-Pt-cRGD upon treatment with
ascorbic acid and caspase-3 in the presence and absence of inhibitor 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino] sulfonylisatin (10 μM). (C) Time-
dependent fluorescence spectra of TPS-DEVD-Pt-cRGD in the presence of caspase-3 in DMSO/PIPES buffer (v/v = 1/199) after the treatment
with ascorbic acid. (D) PL intensity at 480 nm of ascorbic acid (1 mM) pretreated TPS-DEVD-Pt-cRGD (10 μM) upon addition of caspase-3 (200
pM) from 0 to 120 min. Data represent mean values standard deviation, n = 3. (E) PL spectra of ascorbic acid (1 mM) pretreated TPS-DEVD-Pt-
cRGD (10 μM) upon incubation with various amounts of caspase-3 (0, 10, 25, 50, 100, and 200 pM) in DMSO/PIPES buffer (v/v = 1/199) for 60
min. (F) PL intensity at 480 nm for ascorbic acid (1 mM) pretreated TPS-DEVD-Pt-cRGD (10 μM) upon addition of various amounts of caspase-3
in DMSO/PIPES buffer (v/v = 1/199) for 60 min. Data represent mean values standard deviation, n = 3.
change when TPS-DEVD-Pt-cRGD is reduced to the apoptosis
sensor TPS-DEVD. However, strong fluorescence signals are
recorded for ascorbic acid pretreated TPS-DEVD-Pt-cRGD (10
μM) upon further treatment with recombinant human caspase-
3 (200 pM). In addition, most of the fluorescence is readily
competed away by pretreatment of the prodrug with 5-[(S)-
(+)-2-(methoxymethyl)pyrrolidino]sulfonylisatin, a highly spe-
cific inhibitor of caspase-3,16 indicating that specific cleavage of
DEVD from the apoptosis sensor TPS-DEVD is inhibited. The
caspase-3 catalyzed hydrolysis was further confirmed by LC-MS
with the formation of TPS residue with an m/z of 582.659
(Scheme S4, Figures S19−S20, SI). After treatment with
caspase-3, the TPS residue forms nanoparticles with an average
diameter of 109 nm, which explains the solution fluorescence
“turn on” (Figure S21, SI).
The fluorescence change of the TPS-DEVD-Pt-cRGD
solution (10 μM) upon addition of ascorbic acid and caspase-
3 enzyme was also monitored over time in a mixture of
DMSO/PIPES (v/v = 1/199) buffer. As shown in Figure 2C, a
quick fluorescence increase in solution is observed after
incubation with caspase-3. The fluorescence reaches a plateau
in 60 min which is 28-fold higher than the intrinsic emission of
the prodrug (Figure 2D). We further studied the effect of
caspase-3 concentration on the solution emission. Different
concentrations of caspase-3 ranging from 0 to 200 pM were
incubated in DMSO/PIPES (v/v = 1/199) buffer with TPS-
DEVD-Pt-cRGD (10 μM) for 1 h, and the corresponding
spectra are shown in Figure 2E. With increasing concentrations
of caspase-3, the PL intensities are gradually enhanced due to
the increased amount of TPS aggregates formed in aqueous
media. As shown in Figure 2F, the plot of PL intensities at 480
nm against the caspase-3 concentration gives a perfect linear
line (R2 = 0.99), suggesting the possibility of caspase-3
quantification on the basis of the PL intensity changes. The
detection limit for caspase-3 was estimated to be 1 pM on the
basis of the 3×σ method.
To study the selectivity of the prodrug, we incubated
ascorbic acid-pretreated TPS-DEVD-Pt-cRGD (10 μM) with
several proteins, including lysozyme, pepsin, bovine serum
albumin (BSA), and trypsin under identical conditions. As
shown in Figure 3A, only caspase-3 displays a 28-fold
fluorescence increase, whereas the intensities from other
proteins remain low, confirming that DEVD is specifically
recognized and cleaved by caspase-3. In addition, among the
enzymes in the caspase family, only caspase-3 and -7 can cleave
the DEVD peptide (Figure S22, SI). These results demonstrate
that the released TPS-DEVD can be used as a specific indicator
of caspase-3/-7 in the cells. As ascorbic acid and many different
kinds of proteins and enzymes exist inside the cells, we further
obtained the cellular lysate of normal and apoptotic U87-MG
cells, which were pretreated with commonly used cell
apoptosis-inducer staurosporine (STS, 2 μM) to activate the
caspase-3/-7 enzymes in the cells.17 The cell lysates were
directly incubated with TPS-DEVD-Pt-cRGD (5 μM), and the
fluorescence intensity at 480 nm was monitored over time. As
shown in Figure 3B, the fluorescence intensity increases quickly
in a way similar to that of the solution study in Figure 2C.
Meanwhile, the fluorescence intensity at 480 nm shows a
minimum change after incubation with the normal cell lysate
without staurosporine treatment, indicating that the apoptosis
sensor is highly stable upon treatment with cellular proteins
where caspase is not present.
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TPS-DEVD can be used as an indicator of cell apoptosis. To
further evaluate whether the probe can be used to quantify the
efficiency of apoptosis-inducing agents, TPS-DEVD-pretreated
U87-MG cells were treated with different concentrations of
cisplatin, and the fluorescence intensity was studied. As shown
in Figure S24, SI, with increasing concentration of cisplatin
used, the cell fluorescence is progressively intensified, indicating
that our probe can be used for semiquantitative analysis of
apoptosis-related drug efficacy in living cells. Next, we
incubated the prodrug TPS-DEVD-Pt-cRGD with U87-MG
human glioblastoma, MCF-7 breast cancer cells and normal
293T cells. The confocal imaging results are shown in Figure 4.
U87-MG cells with overexpressed αvβ3 integrin on the cellular
membrane were chosen as integrin-positive cancer cells, while
MCF-7 and 293T cells with low αvβ3 integrin expression were
used as the negative controls. Upon incubation with TPS-
DEVD-Pt-cRGD, the fluorescence of U87-MG cells increases
gradually with the cellular apoptotic progress, which reaches a
maximum at 6 h. On the contrary, only weak fluorescence
signals could be found for MCF-7 and 293T cells within the
same period of time. When U87-MG cells were pretreated with
free cRGD and/or caspase-3 inhibitor prior to TPS-DEVD-Pt-
cRGD incubation, the image shows weak fluorescence (Figure
S25, SI). These results clearly demonstrate that TPS-DEVD-Pt-
cRGD not only can be used for targeted drug delivery but also
has the potential for real-time monitoring of caspase-3 activity
in situ. Furthermore, excellent overlap is observed between the
confocal images of the TPS residues and immunofluorescence
signals generated from anti-caspase-3 primary antibody and a
Texas Red-labeled secondary antibody, indicating that the
sensor released from the prodrug is specific for cell apoptosis
imaging (Figure 5).
Figure 3. (A) Plot of (I − I0)/I0 for TPS-DEVD-Pt-cRGD upon
incubation with different proteins for 60 min, where I and I0 are the PL
intensities at protein concentrations of 200 and 0 pM, respectively. (B)
Time-dependent PL intensity change of TPS-DEVD-Pt-cRGD in
apoptotic U87-MG cell lysate (AP-U87-MG) and normal U87-MG
cell lysate (Nor-U87-MG). Data represent mean values
standard
Finally, we studied the relationship between the apoptosis-
induced fluorescence intensity change and the cytotoxicity
profile of the prodrug in U87-MG and MCF-7 cells. After the
cells were incubated with different concentrations of TPS-
DEVD-Pt-cRGD for 6 h, the fluorescence intensities at 480 nm
were monitored upon excitation at 365 nm. As shown in Figure
6A, for U87-MG cells, the fluorescence increases upon
incubation with the prodrug in the concentration from 0 to 5
μM, which is followed by saturation. At each prodrug
concentration, the cytotoxicity of the corresponding cells was
deviation, n = 3.
To explore the capability of using TPS-DEVD-Pt-cRGD as a
targeted drug delivery system and a drug-induced apoptosis
imaging sensor in cancer cells, we first incubated TPS-DEVD-
pretreated U87-MG cells with cisplatin or STS and monitored
their fluorescence changes with confocal microscopy (Figure
S23, SI). It can be seen that strong fluorescence signals are
collected from the cells treated with cisplatin or STS, as both
can activate the caspase activity. These results demonstrate that
Figure 4. (A−D) Real-time CLSM images displaying the apoptotic progress of TPS-DEVD-Pt-cRGD (5 μM) stained U87-MG cells and confocal
images of MCF-7 (E) and 293T (F) cells upon treatment with TPS-DEVD-Pt-cRGD (5 μM) for 6 h. Nuclei were live stained with DRAQ5. (G−L)
Corresponding fluorescence/nucleus overlay images of (A−F), respectively. All images share the same scale bar (20 μm).
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MG cells vs MCF-7 cells, which indicates that the prodrug can
serve as a targeted drug delivery vehicle and for noninvasive
early imaging and semiquantification of its therapeutic response
in situ.
CONCLUSIONS
■
In summary, we report the synthesis and biological application
of a theranostic Pt(IV) prodrug for targeted drug delivery and
early evaluation of its therapeutic response in situ. The prodrug
can be reduced to active Pt(II) inside the cells and
simultaneously release the cell apoptosis sensor on its axial
position. The reduced Pt(II) can induce the apoptosis of the
cancer cell and activate the caspase-3. The activated caspase-3
further cleaves the DEVD sequence of the apoptosis sensor and
triggers the AIE effect of TPS residue, thus enabling early
evaluation of its therapeutic response in cells with high signal-
to-noise ratios. In addition, the apoptosis-induced fluorescence
intensity in the U87-MG cells shows a good correlation with
the prodrug concentration and the cell viability. These results
indicate that the theranostic drug delivery system with a built-in
apoptosis sensor allows one to target the drug delivery and
evaluate the drug therapeutic response quickly, which is
essential to guide therapeutic decisions such as whether the
treatment works well or the therapeutic regimes should stop.
Figure 5. CLSM images of U87-MG cells upon treatment with TPS-
DEVD-Pt-cRGD (5 μM) and caspase-3 antibody (A−C) or TPS-
DEVD-Pt-cRGD (5 μM) in the presence of inhibitor 5-[(S)-(+)-2-
(methoxymethyl)pyrrolidino]sulfonylisatin (5 μM) and caspase-3
antibody (D−F). Green = probe fluorescence; red = immunofluor-
escence signal generated from anti-caspase-3 primary antibody and a
Texas Red-labeled secondary antibody. All images share the same scale
bar (20 μm).
ASSOCIATED CONTENT
* Supporting Information
■
S
Structural characterization data of TPS-CH2N3 and TPS-
DEVD-Pt-cRGD; absorption spectra of TPS-CH2N3 and
TPS-DEVD-Pt-cRGD; particle size distribution; in vitro
cytotoxicity of TPS-DEVD. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
cheliub@nus.edu.sg
tangbenz@ust.hk
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Singapore National Research Foundation (R-
279-000-390-281), the Ministry of Defense (R279-000-340-
232), Singapore-MIT Alliance of Research and Technology
(SMART), JCO (IMRE/12-8P1103), and the Research Grants
Council of Hong Kong (HKUST2/CRF/10 and
N_HKUST620/11) for financial support.
Figure 6. Correlations between cell viability (72 h) determined by
MTT assay and apoptosis-induced fluorescence intensity (6 h) of U87-
MG (A) and MCF-7 (B) cells upon treatment with TPS-DEVD-Pt-
cRGD at different concentrations.
REFERENCES
■
(1) (a) Langer, R. Nature 1998, 392, 5−10. (b) Davis, M. E.; Chen,
Z.; Shin, D. M. Nat. Rev. Drug Discov. 2008, 7, 771−782.
(2) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F.
Acc. Chem. Res. 2011, 44, 1029−1038.
evaluated using a standard MTT assay after overall incubation
for 72 h. As the apoptosis sensor TPS-DEVD itself shows no
obvious cytotoxicity to the cells (Figure S26, SI), the cell
viability change is simply due to the presence of reduced Pt(II)
in the cells. As shown in Figure 6A, it is obvious that the cell
viability is high when the used prodrug concentration is low
and that the cell viability decreases at higher prodrug
concentrations. When the same experiments were conducted
on MCF-7 cells, the trend is not obvious. These results clearly
demonstrate the selective killing effect of the prodrug on U87-
(3) (a) Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. J. Am.
Chem. Soc. 2011, 133, 16680−16688. (b) Maiti, S.; Park, N.; Han, J.
H.; Jeon, H. M.; Lee, J. H.; Bhuniya, S.; Kang, C.; Kim, J. S. J. Am.
Chem. Soc. 2013, 135, 4567−4572. (c) Weinstain, R.; Segal, E.; Satchi-
Fainaro, R.; Shabat, D. Chem. Commun. 2010, 46, 553−555. (d) Zou,
T. T.; Lum, C. T.; Chui, S. S. Y.; Che, C. M. Angew. Chem., Int. Ed.
2013, 52, 2930−2933. (e) Jana, A.; Devi, K. S. P.; Maiti, T. K.; Singh,
N. D. P. J. Am. Chem. Soc. 2012, 134, 7656−7659.
(4) Kuhl, C. K. Radiology 2007, 244, 672−691.
(5) Hickman, J. A. Cancer Metastasis Rev. 1992, 11, 121−139.
2553
dx.doi.org/10.1021/ja411811w | J. Am. Chem. Soc. 2014, 136, 2546−2554

Journal of the American Chemical Society
Article
(6) Schoenberger, J.; Bauer, J.; Moosbauer, J.; Eilles, C.; Grimm, D.
Curr. Med. Chem. 2008, 15, 187−194.
(7) (a) Huang, X.; Swierczewska, M.; Choi, K. Y.; Zhu, L.; Bhirde, A.;
Park, J.; Kim, K.; Xie, J.; Niu, G.; Lee, K. C.; Lee, S.; Chen, X. Angew.
Chem., Int. Ed. 2012, 51, 1625−1630. (b) Hu, M.; Li, L.; Wu, H.; Su,
Y.; Yang, P. Y.; Uttamchandani, M.; Xu, Q. H.; Yao, S. Q. J. Am. Chem.
Soc. 2011, 133, 12009−12020. (c) Boeneman, K.; Mei, B. C.; Dennis,
A. M.; Bao, G.; Deschamps, J. R.; Mattoussi, H.; Medintz, I. L. J. Am.
Chem. Soc. 2009, 131, 3828−3829. (d) Lovell, J. F.; Chan, M. W.; Qi,
Q.; Chen, J.; Zheng, G. J. Am. Chem. Soc. 2011, 133, 18580−18582.
(e) Lin, S. Y.; Chen, N. T.; Sun, S. P.; Chang, J. C.; Wang, Y. C.; Yang,
C. S.; Lo, L. W. J. Am. Chem. Soc. 2010, 132, 8309−8315. (f) Barnett,
E. M.; Zhang, X.; Maxwell, D.; Chang, Q.; Piwnica-Worms, D. Proc.
Natl. Acad. Sci. U.S.A. 2009, 106, 9391−9396. (g) Dai, N.; Guo, J.;
Teo, Y. N.; Kool, E. T. Angew. Chem., Int. Ed. 2011, 50, 5105−5109.
(h) Shen, B.; Jeon, J.; Palner, M.; Ye, D.; Shuhendler, A.; Chin, F. T.;
Rao, J. Angew. Chem., Int. Ed. 2013, 52, 10511−10514.
(8) (a) Shi, H. B.; Kwok, R. T. K.; Liu, J. Z.; Xing, B. G.; Tang, B. Z.;
Liu, B. J. Am. Chem. Soc. 2012, 134, 17972−17981. (b) Shi, H. B.;
Zhao, N.; Ding, D.; Liang, J.; Tang, B. Z.; Liu, B. Org. Biomol. Chem.
2013, 11, 7289−7296.
(9) (a) Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009,
4332−4353. (b) Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Chem. Soc.
Rev. 2011, 40, 5361−5388. (c) Ding, D.; Li, K.; Liu, B.; Tang, B. Z.
Acc. Chem. Res. 2013, 46, 2441−2453.
(10) (a) Wang, D.; Lippard, S. J. Nat. Rev. Drug Discovery 2005, 4,
307−320. (b) Kelland, L. Nat. Rev. Cancer 2007, 7, 573−584.
(11) Seki, K.; Yoshikawa, H.; Shiiki, K.; Hamada, Y.; Akamatsu, N.;
Tasaka, K. Cancer Chemother. Pharmacol. 2000, 45, 199−206.
(12) (a) Butler, J. S.; Sadler, P. J. Curr. Opin. Chem. Biol. 2013, 17,
175−188. (b) Dhar, S.; Liu, Z.; Thomale, J.; Dai, H. J.; Lippard, S. J. J.
Am. Chem. Soc. 2008, 130, 11467−11476. (c) Graf, N.; Lippard, S. J.
Adv. Drug Delivery Rev. 2012, 64, 993−1004. (d) Wang, X. Y.; Guo, Z.
J. Chem. Soc. Rev. 2013, 42, 202−224.
(13) Barnes, K. R.; Kutikov, A.; Lippard, S. J. Chem. Biol. 2004, 11,
557−564.
(14) Liu, J. Z.; Zheng, R. H.; Tang, Y. H.; Haussler, M.; Lam, J. W.
Y.; Qin, A.; Ye, M. X.; Hong, Y. N.; Gao, P.; Tang, B. Z.
Macromolecules 2007, 40, 7473−7486.
(15) (a) Lopez-Flores, A.; Jurado, R.; Garcia-Lopez, P. J. Pharmacol.
Toxicol. Methods 2005, 52, 366−372. (b) Li, J.; Yap, S. Q.; Chin, C. F.;
Tian, Q.; Yoong, S. L.; Pastorin, G.; Ang, W. H. Chem. Sci. 2012, 3,
2083−2087.
(16) Lee, D.; Long, S. A.; Adams, J. L.; Chan, G.; Vaidya, K. S.;
Francis, T. A.; Kikly, K.; Winkler, J. D.; Sung, C. M.; Debouck, C.;
Richardson, S.; Levy, M. A.; DeWolf, W. E.; Keller, P. M.; Tomaszek,
T.; Head, M. S.; Ryan, M. D.; Haltiwanger, R. C.; Liang, P. H.; Janson,
C. A.; McDevitt, P. J.; Johanson, K.; Concha, N. O.; Chan, W.; Abdel-
Meguid, S. S.; Badger, A. M.; Lark, M. W.; Nadeau, D. P.; Suva, L. J.;
Gowen, M.; Nuttall, M. E. J. Biol. Chem. 2000, 275, 16007−16014.
(17) Luhrmann, A.; Nogueira, C. V.; Carey, K. L.; Roy, C. R. Proc.
Natl. Acad. Sci. U.S.A. 2010, 107, 18997−19001.
2554
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