Light-up bioprobe with aggregation-induced emission characteristics for real-time apoptosis imaging in target cancer cells

Article abstract of DOI:10.1039/c3tb21495h

Specific bioprobes that are capable of real-time and targeted monitoring and imaging of cancer cell apoptosis are highly desirable for cancer diagnosis and the evaluation of cancer therapy efficacy. In this work, an asymmetric fluorescent light-up bioprobe with aggregation-induced emission (AIE) characteristics was designed and synthesized by the conjugation of two different hydrophilic peptides, caspase-specific Asp-Glu-Val-Asp (DEVD) and cyclic Arg-Gly-Asp (cRGD), onto a typical AIE luminogen of a tetraphenylsilole (TPS) unit. The asymmetric probe is almost non-emissive in aqueous solution and its fluorescence is significantly switched on in the presence of caspase-3. The fluorescence turn-on is due to the cleavage of the DEVD moiety by caspase-3, and the aggregation of released TPS-cRGD residues, which restricts the intramolecular rotations of TPS phenyl rings and populates the radiative decay channels. Application of the asymmetric light-up probe for real-time targeted imaging of cancer cell apoptosis is successfully demonstrated using integrin α_vβ₃ receptor overexpressing U87MG human glioblastoma cells as an example. The probe shows specific targeting capability to U87MG cancer cells by virtue of the efficient binding between cRGD and integrin α_vβ₃ receptors and is able to real-time monitor and image cancer cell apoptosis in a specific and sensitive manner. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3tb21495h

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Materials Chemistry B
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PAPER
Light-up bioprobe with aggregation-induced
emission characteristics for real-time apoptosis
Cite this: J. Mater. Chem. B, 2014, 2,
imaging in target cancer cells†
231
Dan Ding,a Jing Liang,a Haibin Shi,a Ryan T. K. Kwok,b Meng Gao,c Guangxue Feng,a
a
bd
ac
*
*
Youyong Yuan, Ben Zhong Tang
and Bin Liu
Specific bioprobes that are capable of real-time and targeted monitoring and imaging of cancer cell
apoptosis are highly desirable for cancer diagnosis and the evaluation of cancer therapy efficacy. In this
work, an asymmetric fluorescent light-up bioprobe with aggregation-induced emission (AIE)
characteristics was designed and synthesized by the conjugation of two different hydrophilic peptides,
caspase-specific Asp-Glu-Val-Asp (DEVD) and cyclic Arg-Gly-Asp (cRGD), onto a typical AIE luminogen
of a tetraphenylsilole (TPS) unit. The asymmetric probe is almost non-emissive in aqueous solution and
its fluorescence is significantly switched on in the presence of caspase-3. The fluorescence turn-on is
due to the cleavage of the DEVD moiety by caspase-3, and the aggregation of released TPS-cRGD
residues, which restricts the intramolecular rotations of TPS phenyl rings and populates the radiative
decay channels. Application of the asymmetric light-up probe for real-time targeted imaging of cancer
cell apoptosis is successfully demonstrated using integrin avb3 receptor overexpressing U87MG human
glioblastoma cells as an example. The probe shows specific targeting capability to U87MG cancer cells
by virtue of the efficient binding between cRGD and integrin avb3 receptors and is able to real-time
monitor and image cancer cell apoptosis in a specific and sensitive manner.
Received 23rd October 2013
Accepted 24th October 2013
DOI: 10.1039/c3tb21495h
www.rsc.org/MaterialsB
developments and to provide assessment of the anticancer
activities of clinically available molecular-targeted drugs. Addi-
1 Introduction
tionally, the high accumulation of apoptosis-monitoring probes
in target cancer cells also minimizes the possible toxic side
effects and false positive signals. As a consequence, effective
and sensitive probes with the capability of real-time apoptosis
imaging in target cancer cells are in urgent pursuit. To date,
there have been several strategies for detecting apoptosis at
cellular level.4–10 Among them, the monitoring of cytosolic cas-
pases, which are a family of cysteine proteases and specic
apoptotic mediators, has been demonstrated to be a very
effective strategy to evaluate apoptosis.2c,5–10 These probes are
generally composed of caspase-specic peptide substrates and
uorescent/bioluminescent units including latent uo-
rophores,5 uorescent proteins,6 uorescence resonance energy
transfer pairs,7 donor/quencher pairs,8 D-lucferin or luciferase.9
These uorescent/bioluminescent units respond to cell
apoptosis upon peptide substrate cleavage by caspases.
Although these methods have enabled the detection of caspase
activity at the cellular level, they oen require a complicated
probe design, and suffer from one or more disadvantages, such
as poor cell permeability, aggregation caused quenching (ACQ),
high background signal, or small uorescence/biolumines-
cence signal changes. More importantly, very few probes have
been reported for apoptosis imaging in target cancer cells,
which are all limited to nanoparticle based systems.10
Apoptosis is a mode of programmed cell death in multicellular
organisms.1 As the deregulation of apoptosis can cause cancer
and responses to anticancer therapeutics, real-time apoptosis
imaging at single cancer cellular level will open up new
opportunities in the early prognosis of cancers, tracing of the
cancer progress, as well as the efficacy estimation of new anti-
cancer agents.2 Recently, specic probes for the identication of
target cancer cells among various cancer cell lines or against
normal cells have attracted increasing interest in fundamental
biological studies and clinical diagnosis.3 Therefore, real-time
apoptosis imaging in target cancer cells is of great importance
in offering scientists with sight and insight into specic cancer
aDepartment of Chemical and Biomolecular Engineering, National University of
Singapore, 117576, Singapore. E-mail: cheliub@nus.edu.sg; Fax: +65 67791936; Tel:
+65 65168049
bDepartment of Chemistry and Division of Biomedical Engineering, Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong,
China. E-mail: tangbenz@ust.hk
cInstitute of Materials Research and Engineering, 3 Research Link, 117602, Singapore
dSCUT–HKUST Joint Research Laboratory, Guangdong Innovative Research Team,
State Key Laboratory of Luminescent Materials and Devices, South China University
of Technology, Guangzhou, 510640, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3tb21495h
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Exploration of new probe designs is highly desirable for real- ability to integrin avb3 receptor overexpressing cancer cells and
time apoptosis monitoring in target cancer cells, which will the specic capability of real-time apoptosis sensing and
provide a useful tool for cancer diagnosis and the evaluation of imaging in live cancer cells with high sensitivity. As compared
cancer therapy efficacy.
to our previous light-up bioprobes, the special asymmetric
Recently, we have developed a novel class of organic lumi- probe design is not only able to detect and image live cancer
nogens with a unique aggregation-induced emission (AIE) cell-specic apoptosis, but also achieve improved sensitivity by
signature, which is exactly opposite to the ACQ effect for providing a low background signal.
conventional uorophores.11 The AIE luminogens generally
have rotating units (e.g. phenyl rings) to promote fast discrete
diffusion. In dilute solutions, the AIE luminogens are non-
2 Experimental
2.1 Materials
emissive and in a “turn-off” state due to the fast non-radiative
decay of the excited states arising from the low-frequency
motions of rotating units. In aggregates, the AIE luminogens
luminesce intensively with uorescence “turn-on” via a mech-
anism of the restriction of intramolecular rotations (RIR).12 By
virtue of the AIE phemomenon, the luminogens with AIE
characteristics are attracting increasing attention and have
been widely used for sensing and imaging applications.13 Most
recently, we have developed AIE luminogen based light-up
probes, which have been successfully used for intracellular
protein monitoring in vitro.14 The successful light-up probe
design strategy has motivated us to explore new specic biop-
robes with AIE characteristics for apoptosis imaging in target
cancer cells.
In this contribution, we designed and synthesized a light-up
molecular probe with an AIE signature for real-time apoptosis
imaging in target cancer cells. Two different peptide sequences,
cyclic Arg-Gly-Asp (cRGD) and the acetyl protective N-terminal
Asp-Glu-Val-Asp (Ac-DEVD), were conjugated to each side of the
tetraphenylsilole (TPS) unit to afford the asymmetric bioprobe
of Ac-DEVD-TPS-cRGD (Scheme 1). cRGD and Ac-DEVD peptides
were selected as the biorecognition units because cRGD shows
high affinity to integrin avb3 receptors that are overexpressed in
many cancer cells15 and Ac-DEVD is a peptide substrate that can
be specically cleaved by caspases-3/7.16 We further demon-
strated that the asymmetric AIE bioprobe shows both targeting
The ethylyne-functionalized cyclic RGD (E-cRGD) and the eth-
ylyne-functionalized acetyl protective N-terminal Asp-Glu-Val-
Asp (Ac-DEVD-E) were purchased from GL Biochem Ltd.
Cathepsin B, lysozyme, pepsin, papsin, trypsin, bovine serum
albumin (BSA), copper(II) sulfate, sodium ascorbate, dimethyl
sulfoxide (DMSO), piperazine-N,N0-bis(2-ethanesulfonic acid)
(PIPES), triuoroacetic acid (TFA), penicillin–streptomycin
solution, trypsin-ethylenediaminetetraacetic acid (EDTA) solu-
tion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) were all purchased from Sigma-Aldrich.
Recombinant human caspase-3, caspase-7 and caspase-1 were
customized from R&D Systems. Annexin V-Alexa Fluor 488 was
purchased from Invitrogen. Staurosporine (STS) was purchased
from Biovision. Caspase inhibitor 5-[(S)-(+)-2-(methoxymethyl)-
pyrrolidino]sulfonylisatin was provided by Calbiochem. Cleaved
caspase-3 (Asp175) (5A1E) Rabbit mAb (#9664) was purchased
from Cell Signaling. Mouse anti-rabbit IgG-TR (sc-3917) was
customized from Santa Cruz. Fetal bovine serum (FBS) was
provided by Gibco (Lige Technologies, Switzerland). Tetrahy-
drofuran (THF) was distilled from sodium benzophenone ketyl
under dry nitrogen immediately prior to use. Milli-Q water was
supplied by a Milli-Q Plus System (Millipore Corporation, Bed-
ford, USA). U87MG human glioblastoma cell line, MCF-7
human breast cancer cell line and 293 T normal cell line were
provided by American Type Culture Collection. The bisazido-
functionalized tetraphenylsilole (BATPS) was synthesized
according to the literature.14a
2.2 Characterization
1H and 13C NMR spectra were measured on a Bruker ARX 600
NMR spectrometer. Chemical shis were reported in parts per
million (ppm) referenced with respect to residual solvent
((CD3)2SO ¼ 2.50 ppm or tetramethylsilane Si(CH3)4 ¼ 0 ppm).
The synthesized samples were puried by preparative high
pressure liquid chromatography (HPLC) (Agilent 1100 series).
The analytical LC proles and molecular mass were acquired
using liquid chromatography-ion trap-time-of-ight mass
spectrometry (LCMS-IT-TOF) (Shimadzu). 0.1% TFA/H2O and
0.1% TFA/acetonitrile were used as eluents for all HPLC exper-
iments. The column used for the preparative HPLC was ZOR-
BAX SB-C18, 5 mm (Agilent) with the following ow parameters:
ow rate ¼ 2 mL minꢀ1, 0 min: 20% ACN, 8 min: 50% ACN, 15
min: 100% ACN. The column used for the analytical HPLC was
Luna 5m C18(2) (Phenomenex®) with the following ow
parameters: ow rate ¼ 0.6 mL minꢀ1; 10–100% ACN from
Scheme 1 Synthetic route to Ac-DEVD-TPS-cRGD.
232 | J. Mater. Chem. B, 2014, 2, 231–238
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0.01 min to 13 min. UV-vis absorption spectra were recorded on 1H), 7.74 (s, 1H), 7.69 (s, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.34 (t, J ¼
a Shimadzu UV-1700 spectrometer. Photoluminescence (PL) 5.4 Hz, 1H), 7.08–7.02 (m, 6H), 6.95 (d, J ¼ 7.2 Hz, 2H), 6.92–6.87
spectra were recorded on a Perkin-Elmer LS 55 spectrouo- (m, 6H), 6.76–6.74 (m, 3H), 6.66–6.63 (m, 3H), 5.34 (s, 2H), 5.32
rometer. All PL spectra were measured with an excitation (s, 2H), 4.53–4.49 (m, 1H), 4.45–4.33 (m, 3H), 4.30–4.22 (m, 2H),
wavelength of 360 nm. The average particle size and size 4.19–4.16 (m, 1H), 4.02–3.95 (m, 3H), 3.17–3.14 (m, 2H), 3.00–
distribution of the samples were measured by laser light scat- 2.96 (m, 4H), 2.90–2.87 (m, 2H), 2.81–2.77 (m, 4H), 2.61–2.52
tering (LLS) with a particle size analyzer (90 Plus, Brookhaven (m, 4H), 2.45–2.35 (m, 2H), 2.27–2.23 (m, 2H), 2.13–2.08
Instruments Co. USA) at a xed angle of 90ꢁ at room (m, 2H), 1.83–1.80 (m, 2H), 1.72 (s, 3H), 1.66–1.63 (m, 2H), 1.41–
temperature.
1.38 (m, 2H), 1.30–1.22 (m, 2H), 0.67 (d, J ¼ 6.0 Hz, 6H), 0.66
(s, 6H). 13C NMR (DMSO-d6, 100 MHz): d 174.0, 172.2, 171.8,
171.6, 171.5, 171.1, 170.9, 170.8, 170.6, 170.0, 169.5, 169.3,
156.6, 153.9, 153.9, 143.2, 142.8, 140.7, 140.6, 138.7, 138.3,
137.2, 133.5, 133.4, 129.2, 128.9, 128.49, 127.9, 127.5, 127.3,
127.2, 126.4, 126.1, 123.1, 123.1, 57.8, 54.3, 53.7, 52.4, 52.3, 52.1,
49.7, 49.6, 48.9, 43.1, 36.7, 35.9, 35.6, 35.1, 30.3, 30.0, 28.1, 27.9,
26.9, 25.0, 22.4, 19.0, 17.9, ꢀ4.2. IT-TOF-MS: m/z [M + 2H]2+ calc.
854.36, found 854.85.
2.3 Synthesis, purication and characterization of Ac-DEVD-
TPS-N3
Excess amounts of BATPS (40 mg, 75 mmol) and Ac-DEVD-E
(9.2 mg, 15 mmol) were dissolved in 0.8 mL of a DMSO and THF
mixture (v/v ¼ 1 : 1). The mixture was sonicated to obtain a clear
solution. A click reaction was initiated by the subsequent
addition of 0.2 mL of an aqueous solution containing CuSO4
(1.2 mg, 7.5 mmol) and sodium ascorbate (3 mg, 15 mmol). The
reaction was constantly monitored by HPLC to maximize the
formation of Ac-DEVD-TPS-N3 and minimize the byproduct of
Ac-DEVD-TPS-DVED-Ac by shaking at low temperature (4 ꢁC) for
a short period (12 h). The desired product of Ac-DEVD-TPS-N3
was then puried by HPLC to yield Ac-DEVD-TPS-N3 (5.1 mg,
30% yield) as a ne powder before further characterization by
1H and 13C NMR as well as LC-MS. 1H NMR (DMSO-d6,
600 MHz): d 12.14 (br s, 3H), 8.13 (d, J ¼ 7.2 Hz, 1H), 8.07 (d, J ¼
7.2 Hz, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.85 (d, J ¼ 7.2 Hz, 1H), 7.72
(s, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 7.07 (s, 1H), 7.02–6.99 (m, 3H),
6.90–6.87 (m, 8H), 6.78–6.72 (m, 5H), 6.66–6.65 (m, 3H), 5.31
(s, 2H), 4.43–4.36 (m, 2H), 4.23 (s, 2H), 4.17–4.13 (m, 1H), 3.99–
3.96 (m, 1H), 2.98–2.94 (m, 1H), 2.79–2.75 (m, 1H), 2.56–2.50
(m, 2H), 2.42–2.33 (m, 2H), 2.13–2.05 (m, 2H), 1.96 (s, 3H), 1.85–
1.75 (m, 2H), 1.65–1.59 (m, 1H), 0.64 (d, J ¼ 6.0 Hz, 6H); 13C
NMR (DMSO-d6, 150 MHz): d 206.4, 174.0, 172.2, 171.8, 171.6,
171.0, 170.9, 170.9, 170.1, 169.4, 153.8, 153.8, 143.2, 140.8,
140.7, 138.9, 138.7, 138.3, 138.3, 133.5, 132.9, 129.3, 129.2,
128.5, 128.1, 127.5, 127.5, 127.3, 126.4, 126.4, 126.4, 123.1, 57.7,
53.3, 52.4, 52.0, 51.1, 49.7, 49.5, 40.0, 39.9, 39.8, 39.7, 39.6, 39.5,
39.3, 39.2, 39.0, 35.9, 35.6, 30.6, 30.3, 29.9, 27.8, 26.9, 22.4, 20.4,
19.0, 17.9. IT-TOF-MS: m/z [M + H]+ calc. 1137.46, found
1137.45.
2.5 Enzymatic assays
In general, 2 mL of Ac-DEVD-TPS-N3 or Ac-DEVD-TPS-cRGD in
DMSO solution (1 mM) was rst diluted with 50 mL of caspase-3
assay PIPES buffer (50 mM PIPES, 100 mM NaCl, 1 mM EDTA,
0.1% w/v CHAPS, 25% w/v sucrose, pH ¼ 7.2), which was fol-
lowed by the addition of a predetermined amount of the
recombinant caspase-3 (40 ng mLꢀ1 stock solution in assay
buffer). The reaction mixture was incubated at 37 ꢁC for 30 min,
and was subsequently diluted to a total of 600 mL with Milli-Q
water for PL measurements upon excitation at 360 nm.
2.6 Cell culture
U87MG human glioblastoma cells, MCF-7 human breast cancer
cells and 293 T normal cells were cultured in Dulbecco's
Modied Eagle's Medium (DMEM) containing 10% FBS and 1%
ꢁ
penicillin-streptomycin at 37 C in a humidied environment
containing 5% CO2, respectively. Before the experiments, the
cells were pre-cultured until conuence was reached.
2.7 Apoptosis imaging in target cancer cells
U87MG glioblastoma cells were cultured in confocal imaging
ꢁ
chambers (LAB-TEK, Chambered Coverglass System) at 37 C.
Aer 80% conuence, the medium was removed and the
adherent cells were washed twice with 1 ꢂ PBS buffer. The
Ac-DEVD-TPS-N3 and Ac-DEVD-TPS-cRGD in FBS-free DMEM
medium at a concentration of 5 mM were then added to the
2.4 Synthesis, purication and characterization of Ac-DEVD-
TPS-cRGD
The puried Ac-DEVD-TPS-N3 (5 mg, 4.4 mmol) and E-cRGD chamber, respectively. Aer incubation at 37 ꢁC for 2 h, the cells
(3.7 mg, 6.5 mmol) were dissolved in 0.5 mL of DMSO. CuSO4 were washed three times with 1 ꢂ PBS buffer and then incu-
(0.35 mg, 2.2 mmol) and sodium ascorbate (0.88 mg, 4.4 mmol) bated with staurosporine (STS, 1 mM) in FBS-free DMEM
dissolved in 0.1 mL of water were added to the mixture to medium for 1 h to induce cell apoptosis. The cell monolayer was
initiate the click chemistry. The reaction was allowed to proceed then imaged by confocal laser scanning microscope (CLSM,
at room temperature under shaking for 2 days. The obtained Zeiss LSM 410, Jena, Germany) with imaging soware (Olympus
product was puried by HPLC to yield Ac-DEVD-TPS-cRGD Fluoview FV1000). For all the images, the confocal set up
(4.5 mg, 60% yield) as a ne powder before further character- remained the same, e.g. the pinhole was set at 145 mm (60ꢂ) and
ization with 1H and 13C NMR as well as LC-MS. 1H NMR (DMSO- the gain was set at 1. The uorescent signals from the probes
d6, 600 MHz): d 12.15 (br s, 6H), 8.17–8.14 (m, 2H), 8.08 (d, J ¼ were collected upon excitation at 405 nm (1 mW) with a 505 nm
7.8 Hz, 1H), 8.00 (d, J ¼ 7.8 Hz, 1H), 7.92 (d, J ¼ 7.2 Hz, 1H), 7.89 longpass barrier lter. MCF-7 breast cancer cells and 293 T
(d, J ¼ 8.4 Hz, 1H), 7.86 (d, J ¼ 7.8 Hz, 2H), 7.81 (d, J ¼ 8.4 Hz, normal cells incubated with Ac-DEVD-TPS-cRGD, respectively,
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were also studied following the same procedures. To study the viability was expressed by the ratio of absorbance of the cells
competitive effect of free cRGD peptide on the cellular uptake of incubated with the sample suspension to that of the cells
Ac-DEVD-TPS-cRGD, the U87MG cells were rst incubated with incubated with culture medium only.
10 mM free cRGD in DMEM medium, which was followed by
incubation with Ac-DEVD-TPS-cRGD in DMEM medium at 37 ꢁC
for 2 h before STS was used to induce cell apoptosis.
3 Results and discussion
The acetyl protective N-terminal Asp-Glu-Val-Asp-TPS-azide
2.8 Co-localization studies
probe (Ac-DEVD-TPS-N3) was rst synthesized in a 30% yield
by a copper-catalyzed “click” reaction between a bisazido (BA)-
functionalized TPS (BATPS) and an ethylyne (E)-functional-
ized Ac-DEVD (Ac-DEVD-E) in a DMSO–THF (v/v ¼ 1 : 1)
mixture. Aerwards the Ac-DEVD-TPS-N3 was puried by
HPLC and characterized with 1H and 13C NMR and LC-MS
(Fig. S1 and S2 in the ESI†), further coupling between Ac-
DEVD-TPS-N3 and E-bearing cyclic RGD (E-cRGD) by click
chemistry yielded Ac-DEVD-TPS-cRGD with a 60% yield. The
purity and identity of the Ac-DEVD-TPS-cRGD were also
For co-localization with Annexin V-Alexa Fluor 488, the Ac-
DEVD-TPS-cRGD-treated apoptotic U87MG cells were further
incubated with a mixture of Annexin V-Alexa Fluor 488/FBS-free
DMEM (v/v ¼ 1 : 799) for 20 min at room temperature, which
was followed by washing twice with 1 ꢂ PBS buffer. The cells
were then kept in fresh FBS-free DMEM for CLSM imaging. For
co-localization with active caspase-3 antibody, the Ac-DEVD-
TPS-cRGD-treated apoptotic U87MG cells were rst xed with
3.7% formaldehyde in 1 ꢂ PBS for 30 min at room temperature.
Aer washing twice with 1 ꢂ PBS, and being permeabilized with
0.1% Triton X-100 in 1 ꢂ PBS for 15 min, the cells were subse-
quently blocked with 2% BSA in 1 ꢂ PBS for 30 min and washed
twice with 1 ꢂ PBS. Subsequently, the cells were further incu-
bated with a mixture of cleaved caspase-3 primary antibody/1 ꢂ
PBS (v/v ¼ 1 : 99) for 2 h at room temperature. Aer washing
twice with 1 ꢂ PBS buffer and then incubating with mouse anti-
rabbit IgG-TR (0.8 mg mLꢀ1) in 1 ꢂ PBS for 4 h, the cells were
washed twice with 1 ꢂ PBS before the cell monolayer was
imaged by CLSM.
1
conrmed by H and 13C NMR and LC-MS (Fig. S3 and S4 in
the ESI†).
BATPS, Ac-DEVD-TPS-N3 and Ac-DEVD-TPS-cRGD exhibit
similar UV-vis absorption spectra in a DMSO–water (v/v ¼ 1/
199) mixture with a peak centered at 360 nm (Fig. S5 in ESI†).
The photoluminescence (PL) spectra are shown in Fig. 1A.
BATPS shows high uorescence in a DMSO–water (v/v ¼ 1/199)
mixture with a quantum yield (F) of 0.17, measured using
quinine sulfate in 0.1 M H2SO4 as the standard.17 In compar-
ison, Ac-DEVD-TPS-N3 is weakly uorescent (F ¼ 0.01) and Ac-
DEVD-TPS-cRGD is almost non-emissive (F ¼ 0.001) in the
same medium. The high uorescence of BATPS is because the
hydrophobic BATPS molecules form nanoaggregates in
aqueous medium with an average size of ꢃ95 nm measured by
laser light scattering (LLS) and atomic force microscopy (AFM)
(Fig. S6 in the ESI†). The conjugation of the Ac-DEVD peptide
with TPS, however, leads to Ac-DEVD-TPS-N3 with improved
water-solubility relative to that of BATPS. Further coupling of
the cRGD peptide endows the Ac-DEVD-TPS-cRGD with good
water-solubility, which is further conrmed by the undetect-
able LLS signal. The high brightness for the BATPS aggregates
and the almost zero uorescence for the Ac-DEVD-TPS-cRGD
molecular probe agree with the AIE mechanism. Due to the
propeller-shaped structure of TPS,12 the dynamic rotations of
the phenyl rings non-radiatively deactivated their excited
states in solution; in the aggregate state, the RIR opens the
2.9 Real-time imaging of cancer cell apoptosis
U87MG glioblastoma cells were cultured in confocal imaging
ꢁ
chambers at 37 C. Aer 80% conuence, 5 mM Ac-DEVD-TPS-
cRGD in FBS-free DMEꢁM medium was added to the chamber.
Aer incubation at 37 C for 2 h, the cells were washed three
times with 1 ꢂ PBS buffer. Subsequently, the confocal imaging
chambers were placed on the CLSM platform, which was fol-
lowed by the addition of STS (1 mM) in FBS-free DMEM medium.
The cell monolayer was then imaged immediately by CLSM at
the designated time intervals. All the images were taken under
the same imaging conditions with excitation at 405 nm and a
505 nm longpass barrier lter.
2.10 Cytotoxicity study
The cytotoxicities of Ac-DEVD-TPS-N3 and Ac-DEVD-TPS-cRGD radiative decay channel. The extremely low background uo-
against U87MG glioblastoma cells were evaluated by MTT assay. rescence signal makes the Ac-DEVD-TPS-cRGD probe prom-
Briey, U87MG cells were seeded in 96-well plates (Costar, IL, ising to serve as an effective light-up probe with high
USA) at an intensity of 4 ꢂ 104 cells per mL. Aer 24 h incu- sensitivity.
bation, the cells were exposed to a series of doses of Ac-DEVD-
The principle of the probe Ac-DEVD-TPS-cRGD for apoptosis
ꢁ
TPS-N3 or Ac-DEVD-TPS-cRGD at 37 C. Aer 48 h incubation, imaging in target cancer cells is illustrated in Scheme 2. By
the wells were washed twice with 1 ꢂ PBS and 100 mL of freshly virtue of the specic binding between cRGD peptide and
prepared MTT (0.5 mg mLꢀ1) solution in culture medium was integrin avb3 receptors, the Ac-DEVD-TPS-cRGD should be
added into each well. The MTT medium solution was carefully favorably internalized by integrin avb3 receptor-overexpressed
removed aer 3 h incubation in the incubator. DMSO (100 mL) cancer cells over other cells with low receptor expression on the
was then added into each well and the plate was gently shaken cell membrane. As Ac-DEVD-TPS-cRGD is highly water-soluble
for 10 minutes at room temperature to dissolve all the precipi- and shows almost no uorescence in aqueous solution, it is
tates that had formed. The absorbance of MTT at 570 nm was hypothesized that the intracellular Ac-DEVD-TPS-cRGD inter-
monitored by the microplate reader (Genios Tecan). Cell nalized by healthy cancer cells without apoptosis can still
234 | J. Mater. Chem. B, 2014, 2, 231–238
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Scheme 2 The principle of apoptosis imaging in target cancer cell
based on Ac-DEVD-TPS-cRGD.
To test our hypothesis, in vitro enzymatic assays with
recombinant caspase-3, one of the key mediators of cell
apoptosis in the caspase family,18 were rst carried out. Caspase-
3 (80 ng mLꢀ1) was mixed with Ac-DEVD-TPS-N3 (3.3 mM) or Ac-
DEVD-TPS-cRGD (3.3 mM) in piperazine-N,N0-bis(2-ethane-
sulfonic acid) (PIPES) buffer (pH ¼ 7.2), which was then incu-
bated at 37 ꢁC for 30 min. The corresponding PL spectra are
shown in Fig. 1B. High uorescent signals ranging from 400 to
650 nm are observed for both probes upon treatment with cas-
pase-3. Moreover, most of the uorescence cannot be observed
by pretreatment of the two probes with 5-[(S)-(+)-2-(methox-
ymethyl)pyrrolidino] sulfonylisatin, a highly specic inhibitor
for caspase-3.19 The caspase-3-catalyzed hydrolysis of Ac-DEVD-
TPS-cRGD was further veried by LC-MS (Fig. S7 in ESI†), which
conrms that the uorescence turn-on is through the specic
cleavage of Ac-DEVD from the probe by caspase-3. It is note-
worthy that the uorescence intensity of the residual TPS-cRGD
nanoaggregates (average diameter of ꢃ43 nm, Fig. S8A and S8B
in ESI†, F ¼ 11% in water) is about 70% of that of the TPS-N3
nanoaggregates (average diameter of ꢃ94 nm, Fig. S8C in ESI†),
because the cRGD peptide is hydrophilic, and as a consequence
the TPS-cRGD fragment is more hydrophilic than TPS-N3.
We next optimized the concentration of recombinant cas-
pase-3 for the complete digestion of Ac-DEVD-TPS-cRGD.
Fig. 1C shows the PL spectra of Ac-DEVD-TPS-cRGD (3.3 mM)
aer incubation with the enzyme in the concentration range
from 0 to 160 ng mLꢀ1 in PIPES buffer at 37 ꢁC for 30 min. With
Fig. 1 (A) Photoluminescence (PL) spectra of BATPS, Ac-DEVD-TPS-
N3 and Ac-DEVD-TPS-cRGD in DMSO–water (v/v ¼ 1 : 199). Inset: the
corresponding photographs taken under illumination of a UV lamp. (B)
PL spectra of Ac-DEVD-TPS-N3 and Ac-DEVD-TPS-cRGD upon
treatment with caspase-3 in the presence and absence of caspase-3
inhibitor (Inh). (C) PL spectra of Ac-DEVD-TPS-cRGD treated with
various amounts of caspase-3 in PIPEs buffer at 37 ꢁC for 30 min. (D)
Plot of (I ꢀ I0)/I0 versus the caspase-3 concentration in PIPEs buffer. I
and I0 are the PL intensities of the assay in the presence and absence of
caspase-3, respectively. Inset: the photograph taken under illumina-
tion of a UV lamp for Ac-DEVD-TPS-cRGD upon treatment with 80 ng
mLꢀ1 of caspase-3 for 30 min. (E) PL intensity changes of Ac-DEVD-
TPS-cRGD upon incubation with and without caspase-3 from 0 to 30
min. (F) Plot of (I ꢀ I0)/I0 versus a variety of proteins, where I and I0 are
the PL intensities at protein concentrations of 80 and 0 ng mLꢀ1
,
respectively. [BATPS] ¼ [Ac-DEVD-TPS-N3] ¼ [Ac-DEVD-TPS-cRGD]
¼ 3.3 mM and lex ¼ 360 nm for (A–F); [caspase-3] ¼ 80 ng mLꢀ1 for
(B and E); [inhibitor] ¼ 10 mM for (B).
maintain the complete uorescence “off” state. On the other the increasing concentration of caspase-3, the probe emission
hand, when the probe is internalized into apoptotic cancer cells gradually increases. A ꢃ70-fold increase in solution uores-
or healthy cancer cells upon drug induced apoptosis, the acti- cence is observed when the probe is treated with 160 ng mLꢀ1 of
vated caspase enzymes should specically cleave the Ac-DEVD caspase-3, as compared to that of the probe alone. As the PL
moiety from the probe, leading to the release of TPS-cRGD. The intensity of the probe is close to saturation upon treatment with
amphiphilic TPS-cRGD molecules will subsequently turn on 80 ng mLꢀ1 of caspase-3, the condition is used for the following
their uorescence upon binding to cell components or form enzymatic experiments. In addition, the PL intensities of the
nanoaggregates in situ due to the RIR. These intermolecular probes aer treatment with caspase-3 increase linearly when
interactions also help to retain the AIE probes inside the cancer the enzyme concentration is in the range of 0 to 80 ng mLꢀ1
cells, which afford good contrast for the real-time imaging of (Fig. 1D), indicating the possible use of Ac-DEVD-TPS-cRGD for
apoptotic progress.
caspase-3 quantication.
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Paper
Fig. 1E illustrates the enzyme kinetic study from monitoring
the PL intensity variations of Ac-DEVD-TPS-cRGD (3.3 mM) upon
incubation with recombinant caspase-3 (80 ng mLꢀ1) in PIPES
ꢁ
buffer at 37 C within a 30 min duration. Aer enzyme treat-
ment, a signicant increase in solution uorescence over the
background is observed over time. In comparison, no obvious
uorescence change is detected in the absence of caspase-3.
These results also conrm the specic cleavage of Ac-DEVD-
TPS-cRGD by caspase-3. In addition, to further demonstrate the
probe selectivity, the same amount of Ac-DEVD-TPS-cRGD was
treated with different proteins, including cathepsin B, lyso-
zyme, pepsin, papsin, trypsin, bovine serum albumin (BSA),
caspase-1 and caspase-7 under the same experimental condi-
tions. As shown in Fig. 1F, caspase-3 and caspase-7 exhibit far
larger changes in (I ꢀ I0)/I0, as compared to other proteins,
revealing that Ac-DEVD-TPS-cRGD is a unique probe for the
specic recognition of caspase-3/7.
Apoptosis imaging in live target cancer cells based on Ac-
DEVD-TPS-cRGD was studied with confocal laser scanning
microscopy (CLSM). U87MG human glioblastoma cells (integrin
avb3 receptor overexpression), MCF-7 human breast cancer cells
(low integrin avb3 receptor expression) and 293 T normal cells
(low integrin avb3 receptor expression) were chosen to demon-
strate the utility of Ac-DEVD-TPS-cRGD in the apoptosis
imaging of target cancer cells.20 Aer incubating the cells with
ꢁ
the probe at 37 C for 2 h, staurosporine (STS, 1 mM), a drug
known for inducing cell apoptosis,21 was added, and the cells
were further incubated for 1 h before CLSM imaging. As shown
in Fig. 2A–C, for the Ac-DEVD-TPS-cRGD-treated U87MG cancer
cells, the cell apoptosis induced by STS can switch on the
uorescence within the cells and the uorescent signals are
signicantly blocked when STS-induced U87MG cells are pre-
treated with the specic caspase inhibitor, 5-[(S)-(+)-2-
(methoxymethyl)pyrrolidino] sulfonylisatin prior to probe
incubation. These results substantiate that the specic recog-
nition and cleavage of Ac-DEVD-TPS-cRGD by caspases occurs
within live cancer cells. It is noteworthy that the uorescence
intensity of Ac-DEVD-TPS-cRGD in healthy U87MG cancer cells
is negligible (Fig. 2A), thanks to the good water-solubility of the
probe. Aer STS induced cell apoptosis, the activated caspase
enzymes trigger the digestion of Ac-DEVD from the probe, and
the amphiphilic TPS-cRGD residues (Fig. S7 in ESI†) light up in
the cells. It is important to note that the uorescence contrast and Ac-DEVD-TPS-cRGD at 37 C for 2 h, respectively, the cells
between the images shown in Fig. 2A and B is very sharp, which were lysed and the cell lysates were analyzed by HPLC. As shown
clearly indicates that Ac-DEVD-TPS-cRGD is very sensitive to the in Fig. S10 in ESI,† both probes possess good stability in a
apoptosis imaging of live target cancer cells with minimum cellular environment for 2 h and ꢃ1.9-fold more Ac-DEVD-TPS-
background signal. In contrast, obvious green uorescence is cRGD can be internalized into U87MG cells as compared to that
observed when healthy U87MG cancer cells were incubated with for Ac-DEVD-TPS-N3. This should be due to the specic inter-
Ac-DEVD-TPS-N3 (Fig. S9A in ESI†), which highlights the actions between cRGD in the probe and the integrin avb3
contribution of the cRGD conjugation in reducing the probe receptors on the U87MG cell membrane, which favor a more
background signal in cells. The uorescence change in Fig. S9B efficient cellular uptake of Ac-DEVD-TPS-cRGD. In addition, as
and S9C in the ESI† further conrms the function of the cas- compared to Ac-DEVD-TPS-cRGD-treated apoptotic U87MG
Fig. 2 CLSM images of healthy (A) and apoptotic U87MG cells (B) upon
treatment with Ac-DEVD-TPS-cRGD; STS-induced U87MG cells pre-
treated with caspase inhibitor before Ac-DEVD-TPS-cRGD staining
(C); U87MG cells pretreated with 10 mM cRGD followed by staining
with Ac-DEVD-TPS-cRGD and STS induced cell apoptosis (G); healthy
(H) and apoptotic MCF-7 cells (I) treated with Ac-DEVD-TPS-cRGD.
(D–F) and (J–L) are the corresponding fluorescence/transmission
overlay images of (A–C) and (G–I), respectively. [Ac-DEVD-TPS-cRGD]
¼ 5 mM, [STS] ¼ 1 mM, [inhibitor] ¼ 10 mM. Scale bar: 30 mm for all the
images.
ꢁ
pases and the inhibitor.
cells (Fig. 2B), the blocking of integrin avb3 receptors on the
To demonstrate the targeting ability of Ac-DEVD-TPS-cRGD U87MG cell membrane with free cRGD peptides leads to
to integrin avb3 receptor overexpressing cancer cells, a set of cell signicantly reduced uorescent signals aer inducing the
experiments were conducted using U87MG cells as an example. apoptosis (Fig. 2G). Quantitative study using Image Pro Plus
Aer incubating the healthy U87MG cells with Ac-DEVD-TPS-N3 soware indicates that the average uorescence intensity from
236 | J. Mater. Chem. B, 2014, 2, 231–238
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Journal of Materials Chemistry B
each cell in Fig. 2B is ꢃ1.8 times higher than that in Fig. 2G.
To evaluate the capability of Ac-DEVD-TPS-cRGD in the real-
These results manifest that the U87MG cell uptake of Ac-DEVD- time imaging of the apoptotic process in live cancer cells,
ꢁ
TPS-cRGD is suppressed by free cRGD treatment, verifying that U87MG cells were incubated with Ac-DEVD-TPS-cRGD at 37 C
the cRGD peptide/integrin interaction promotes the probe for 2 h. Aer the cells were further treated with STS, they were
internalization into U87MG cancer cells.
immediately imaged by CLSM. Fig. 3 displays the real-time
MCF-7 cells and 293 T cells with low integrin avb3 receptor CLSM images recorded within a 1 h duration. The uorescence
expression were further utilized as controls to study the intensity inside the U87MG cells gradually increases over time
specic targeting ability of Ac-DEVD-TPS-cRGD to integrin avb3 along with the cellular apoptotic progress, and the saturation is
receptor-overexpressing cancer cells. Fig. 2H and I show the observed at 45 min. These results substantiate that Ac-DEVD-
Ac-DEVD-TPS-cRGD-stained MCF-7 cells before and aer STS TPS-cRGD can serve as an effective light-up probe for the real-
induced cell apoptosis, respectively. Almost no signal is time imaging of cancer cell apoptosis. Additionally, the low
detected in the Ac-DEVD-TPS-cRGD-treated healthy MCF-7 cytotoxicity of Ac-DEVD-TPS-cRGD has also been demonstrated,
cells. Moreover, aer STS induced cell apoptosis, although which makes it a safe probe for bioimaging applications
green uorescence from Ac-DEVD-TPS-cRGD-treated apoptotic (Fig. S13 in ESI†).
MCF-7 cells is observed, their intensities are ꢃ2.2 times lower
(analyzed by Image Pro Plus soware) than that from the Ac-
DEVD-TPS-cRGD-treated apoptotic U87MG cells (Fig. 2B).
4 Conclusions
Similarly, low uorescence is observed from Ac-DEVD-TPS-
cRGD-treated apoptotic 293 T normal cells (Fig. S11 in ESI†).
These results conrm the specic targeting ability of Ac-DEVD-
TPS-cRGD to integrin avb3 receptor-overexpressing cancer
cells, which not only favors apoptosis imaging in target cancer
cells, but is also benecial to achieving high contrast in
imaging.
In summary, we developed an asymmetric AIE bioprobe by the
conjugation of Ac-DEVD and cRGD peptides onto a TPS lumi-
nogen. Opposite to the high uorescence of azide functional-
ized TPS aggregates in aqueous media, the probe of Ac-DEVD-
TPS-cRGD is dissolved as molecular species with almost no
emission in aqueous solution. Thanks to the AIE feature of the
TPS-cRGD resides, the probe is able to turn on its uorescence
In addition, Ac-DEVD-TPS-cRGD-treated apoptotic U87MG
when the activated caspases cleave the Ac-DEVD peptide from
cells were co-stained with commercially available Annexin
the probe. Cancer cellular apoptosis imaging studies, using
integrin avb3 receptor-overexpressed U87MG cancer cells as an
example, demonstrate that Ac-DEVD-TPS-cRGD has a specic
targeting ability to U87MG cancer cells and can serve as a safe
and efficient uorescent light-up probe for real-time apoptosis
imaging. This, in combination with the lower background
signal of the probe to that of the Ac-DEVD-TPS-N3, reveals the
dual functions that the cRGD played in the probe design,
V-Alexa Fluor 488 (ref. 22) and anti-caspase-3 primary antibody/
Texas Red-labeled secondary antibody,23 respectively. As shown
in Fig. S12A–C in ESI,† the signal of Annexin V-Alexa Fluor 488 is
observed on the cell surface, whereas Ac-DEVD-TPS-cRGD
displays high uorescence in the cytoplasma. Moreover, the
green uorescence from Ac-DEVD-TPS-cRGD and the red uo-
rescence from Texas Red co-localize well inside the cells
(Fig. S12D–F in ESI†). This data clearly conrms that Ac-DEVD-
namely, the targeting effect and higher contrast or improved
TPS-cRGD is indeed an efficient and specic probe for the
sensitivity by providing a low background signal.
detection of caspase activity and apoptosis imaging in live
cancer cells.
Acknowledgements
We thank the Singapore National Research Foundation (R-279-
000-390-281), Singapore-MIT Alliance for Research and Tech-
nology (SMART) Innovation Grant (R279-000-378-592), the
Institute of Materials Research and Engineering of A-star, Sin-
gapore (IMRE12-8P1103), the Research Grants Council of Hong
Kong (HKUST2/CRF/10and N_HKUST620/11), and Guangdong
Innovative
Research
Team
Program
of
China
(20110C0105067115) for nancial support.
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