Real-time monitoring of cell apoptosis and drug screening using fluorescent light-up probe with aggregation-induced emission characteristics

Article abstract of DOI:10.1021/ja3064588

Real-time monitoring of cell apoptosis could provide valuable insights into early detection of therapy efficiency and evaluation of disease progression. In this work, we designed and synthesized a new live-cell-permeable, fluorescent light-up probe for real-time cell apoptosis imaging. The probe is comprised of a hydrophilic caspase-specific Asp-Glu-Val-Asp (DEVD) peptide and a hydrophobic tetraphenylethene (TPE) unit, a typical fluorogen with aggregation-induced emission characteristics. In aqueous solution, the probe is almost nonfluorescent but displays significant fluorescence enhancement in response to caspase-3/-7, which are activated in the apoptotic process and able to cleave the DEVD moieties. This fluorescence "turn-on" response is ascribed to aggregation of cleaved hydrophobic TPE residues, which restricts the intramolecular rotations of TPE phenyl rings and populates the radiative decay channels. The light-up nature of the probe allows real-time monitoring of caspase-3/-7 activities both in solutions and in living cells with a high signal-to-noise ratio. The probe provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated drug efficacy.

Full text of DOI:10.1021/ja3064588

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Article
Real-time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent
Light-up Probe with Aggregation-Induced Emission Characteristics
Haibin Shi, Ryan T.K. Kwok, Jianzhao Liu, Bengang Xing, Ben Zhong Tang, and Bin Liu
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 08 Oct 2012
Downloaded from http://pubs.acs.org on October 14, 2012
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Realꢀtime Monitoring of Cell Apoptosis and Drug
Screening Using Fluorescent Lightꢀup Probe with
AggregationꢀInduced Emission Characteristics
Haibin Shi,†,≠ Ryan T. K. Kwok#,≠, Jianzhao Liu,# Bengang Xing, Ben Zhong Tang,#,§,* and Bin Liu†,§,*
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†Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of
Singapore, Singapore, 117576,
#Department of Chemistry, Institute for Advanced Study, Division of Biomedical Engineering, State
Key Laboratory of Molecular Neuroscience and Institute of Molecular Functional Materials, The Hong
Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
§Institute of Materials Research Engineering, 3 Research Link, 117602, Singapore
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences
Nanyang Technological University, 637371, Singapore
* Corresponding author (Eꢀmail: cheliub@nus.edu.sg, tangbenz@ust.hk)
≠ Haibin Shi and Ryan Kwok contributed equally to this work.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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ABSTRACT: Realꢀtime monitoring of cell apoptosis could provide valuable insights into early
detection of therapy efficiency and evaluation of disease progression. In this work, we designed and
synthesized a new liveꢀcell permeable, fluorescent lightꢀup probe for realꢀtime cell apoptosis imaging.
The probe is comprised of a hydrophilic caspaseꢀspecific AspꢀGluꢀValꢀAsp (DEVD) peptide and a
hydrophobic tetraphenylethene (TPE) unit, a typical fluorogen with aggregationꢀinduced emission (AIE)
characteristics. In aqueous solution, the probe is almost nonꢀfluorescent but displays significant
fluorescence enhancement in response to caspaseꢀ3/7, which are activated in the apoptotic process and
able to cleave the DEVD moieties. This fluorescence “turnꢀon” response is ascribed to aggregation of
cleaved hydrophobic TPE residues, which restricts the intramolecular rotations of TPE phenyl rings and
populates the radiative decay channels. The lightꢀup nature of the probe allows realꢀtime monitoring of
caspaseꢀ3/7 activities both in solutions and in living cells with a high signal to noise ratio. The probe
provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosisꢀassociated drug
efficacy.
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KEYWORDS: AggregationꢀInduced Emission (AIE) • Cell Apoptosis • Lightꢀup Probe • Caspases •
Realꢀtime Imaging • Enzyme Activity • Drug Screening
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INTRODUCTION Apoptosis, or programmed cell death, is an important and active regulatory
pathway of cell growth and proliferation.1,2 Deregulation of the cell apoptosis can ultimately lead to
many diseases, such as cancers, neurodegenerative diseases, autoimmune diseases, atherosclerosis,
myocardial infarction, and many others.3,4 Realꢀtime imaging the progress of apoptosis in living
organisms therefore has great implications for early diagnosis of diseases, evaluation of disease
progression and therapy efficiency, and screening of apoptosisꢀrelated drugs.5
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Cells undergoing apoptosis generally show characteristic biochemical changes, which include
translocation of phosphatidylserine (PS) from the cytoplasmic leaflet to the extracellular leaflet of the
plasma membrane, DNA fragmentation, protease activation and so on.6 So far, a number of strategies
have been attempted to monitor the apoptosis progress.7ꢀ10 One common approach is to target the
negatively charged PS with annexin V by taking advantage of the specific binding between them.7,11
However, false positive results have been reported because PS exposure also occurs in other biological
processes like necrosis.12 Laddering of DNA fragmentation is another widely used method for apoptosis
monitoring. It requires multiple reaction steps and the internucleosomal DNA cleavages are not always
associated with apoptosis.13,14 To overcome the limitations of these methods, assays for more specific
key players in the apoptosis such as the caspases have recently received considerable attentions.
Caspases are a family of intracellular cysteine proteases that play critical roles in the initiation and
execution of apoptosis.4,15ꢀ17 Among them, caspaseꢀ3 has been identified as a key mediator of cell
apoptosis and it becomes an attractive and unambiguous target for apoptosis imaging. So far, two major
groups of fluorescent probes have been developed to monitor caspase activities.18ꢀ23 The first group
refers to fluorogenic probes containing caspaseꢀspecific peptide substrates and luciferin or luciferase19
or latent fluorophores.20 Fluorogenic peptide substrates containing Cꢀterminally capped coumarin
derivative (i.e., 7ꢀaminoꢀ4ꢀtrifluoromethylcoumarin (AFC)) are arguably the most useful probes for
substrate specificity profiling experiments, as the cleavage at the amide bond between the peptide and
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the coumarin moiety will release the fluorescent coumarin.20 However, these fluorogenic probes have
not been effective for cell apoptosis imaging due to high fluorescence background and poor cell
permeability. The second group is dual labeled probes, which rely on functionalization of the caspase
specific peptide substrates with donor/quencher pairs,18,21,22 or fluorescence resonance energy transfer
(FRET) pairs on both ends.23 The peptide cleavage by caspases can result in separation of dye/quencher
pair or loss of FRET, which enables the caspase activities to be evaluated via the changes in
fluorescence intensity and wavelength. Although the dual labeled probes have overcome some
disadvantages of fluorogenic probes, they require more complicated synthetic steps, and the background
signal is highly dependent on the quenching efficiency or FRET efficiency. Given the above
considerations, it is highly desirable to develop much simpler, noninvasive and specific probes with
high signal to noise ratio for realꢀtime monitoring of cell apoptosis and direct evaluation of new
apoptosisꢀrelated drugs in living cells.
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Aggregationꢀinduced emission (AIE) is a unique photophysical phenomenon that was first reported in
2001.24 Propellerꢀshaped fluorogens, such as tetraphenylethene (TPE), are nonemissive when
molecularly dissolved in solution but induced to emit efficiently by aggregate formation.25 We have
rationalized the AIE mechanism as restriction of intramolecular rotations (RIR) in the aggregate state.26
To date, a variety of fluorogens with AIE characteristics have been developed for applications in
chemical sensors,25b,27,28 biological imaging29 and optoelectronic devices.26a,30 Of particular importance
is that the AIE phenomenon has been demonstrated useful in monitoring activities of various enzymes,
such as trypsin,31 acetylcholinesterase (AChE)32 and alkaline phosphatase.33 These assays rely on
weakly fluorescent AIE fluorogens in solutions to interact with oppositely charged chemical or protein
substrates to yield fluorescent complexes, which then release the AIE fluorogens back to the solutions
upon enzyme digestion and lead to fluorescence “turnꢀoff”. Although many efforts have also been made
to develop AIE based fluorescence “turnꢀon” assays for enzyme activity studies,34 multiple reagents or
chemical reactions have to be involved, which make them also not suitable for cellular based studies.
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In this contribution, we designed and synthesized a probe via conjugation between a hydrophilic
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DEVD peptide sequence and a hydrophobic AIE fluorogen. The probe is highly waterꢀsoluble and
nonfluorescent in aqueous environment. The specific cleavage of DEVD by caspases induces
aggregation of the hydrophobic AIE residues and thus enhances the fluorescence signal output. This
probe is capable of detecting caspaseꢀ3/7 activities both in solution and in living cells. By monitoring
the activity of caspaseꢀ3 in living cells, we further demonstrated that our probe could be used for realꢀ
time apoptosis imaging and in situ apoptosisꢀrelated drug screening with high fluorescence contrast.
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RESULTS AND DISCUSSION
Design Principle of AIE-based Probe. TPE is a classic AIE fluorogen, which emits strongly in
aggregate state but shows very weak fluorescence in dilute solutions.25 This is due to the propellerꢀ
shaped structure of TPE, and the dynamic rotations of the phenyl rings nonꢀradiatively deactivated their
excited states in solution. In the aggregate state, the restriction of the intramolecular rotations (RIR) due
to the physical constraint in the aggregates opens the radiative decay channel.26 Our design rationale is
illustrated in Scheme 1. The acetyl protective Nꢀterminal AspꢀGluꢀValꢀAspꢀLysꢀTPE probe (Acꢀ
DEVDKꢀTPE) is made of three components: (1) a DEVD sequence that can be specifically cleaved by
caspaseꢀ3/7 and used to endow the probe with water solubility; (2) an azideꢀfunctionalized TPE
fluorogen with modulated fluorescence on/off upon external stimuli; and (3) an alkyneꢀfunctionalized
lysine derivative as a linker to connect DEVD and TPE moieties. AcꢀDEVDKꢀTPE is highly waterꢀ
soluble and displays very weak fluorescence in aqueous media due to the consumption of excitonic
energy by the active intramolecular rotations.24,25 It is hypothesized that once the probe is internalized
into apoptotic cells, the DEVD moiety will be specifically cleaved between Asp(D) and Lys(K) by
caspaseꢀ3/7, leading to the release of lysineꢀconjugated TPE (KꢀTPE). As KꢀTPE is hydrophobic,
molecular aggregation will lead to fluorescence turn on according to the AIE mechanism.24,25 Therefore,
the probe provides a good opportunity for realꢀtime imaging of apoptosis process.
Scheme 1. Illustration of Ac-DEVDK-TPE for Caspase Activity Study
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“OFF”
“ON”
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Scheme 2. Synthetic Route to TPE-N3
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Synthesis and Characterization of Probe Ac-DEVDK-TPE. The synthesis of AcꢀDEVDKꢀTPE
probe involves both solutionꢀ and solidꢀphase chemistry as described in Schemes 2 and 3. Azideꢀ
functionalized tetraphenylethene (3, TPEꢀN3) was synthesized in four steps with a 45% total yield.
Detailed synthesis and characterization of TPEꢀN3 and the intermediates are shown in the experimental
section or supporting information (SI) (SI Figures S1−S6). The alkyneꢀbearing AcꢀDEVDK (Acꢀ
DEVDKꢀA) peptide was prepared by standard solidꢀphase Fmoc peptide chemistry.18d In brief,
deprotection of Fmoc group with 20% piperidine in DMF yielded amineꢀfunctionalized rink amide resin,
which was coupled to the first amino acid, alkyneꢀfunctionalized lysine, to afford the lysineꢀbearing
resin. Following the same procedure, four different amino acids (Asp, Val, Glu and Asp) were
subsequently incorporated onto the resin by standard coupling reactions. Deprotection of the amine
groups, followed by capping with acetic anhydride and cleaving from resin gave the product Acꢀ
DEVDKꢀA. It was further purified by HPLC and characterized with LCꢀMS. Subsequent coupling
between TPEꢀN3 and AcꢀDEVDKꢀA via Cu(I)ꢀcatalyzed “Click” reaction using CuSO4/sodium
ascorbate as the catalyst and DMSO/H2O as the solvent afforded the probe AcꢀDEVDKꢀTPE in 70%
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yield after HPLC purification. The purity and identity of the probe have been fully characterized by
analytical HPLC, NMR and HRꢀMS (SI Figures S7 and S8).
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Scheme 3. Solid-phase Synthesis of Ac-DEVDK-TPE
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H
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FmocHN
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Rink-amide Resin
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COOH
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NH2
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O
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COOtBu
COOH
COOtBu
COOH
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NH2
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Ac-DEVDK-TPE
Reagents and Conditions: (a) 20% piperidine in DMF; (b) FmocꢀLys(alkyne)ꢀCOOH, HBTU, HOBt, DIEA,
DMF; (c) 20% piperidine in DMF; (d) i. FmocꢀAsp(OtBu)ꢀOH, HBTU, HOBt, DIEA, DMF; ii. 20% piperidine in
DMF; (e) i. FmocꢀValꢀOH, HBTU, HOBt, DIEA, DMF; ii. 20% piperidine in DMF; (f) i. FmocꢀGlu(OtBu)ꢀOH,
HBTU, HOBt, DIEA, DMF; ii. 20% piperidine in DMF; (g) i. FmocꢀAsp(OtBu)ꢀOH, HBTU, HOBt, DIEA,
DMF; ii. 20% piperidine in DMF; iii. acetic anhydride, DIEA; (h) TFA/TIS/H2O (v/v/v = 95:2.5:2.5), 3 h; (i)
TPEꢀN3 (1.0 eqv), CuSO4 (0.2 eqv), sodium ascorbate (0.4 eqv) in DMSO/H2O (1:1), 24 h. Abbreviation: HBTU
(OꢀbenzotriazoleꢀN,N,N’,N’ꢀtetramethyluronium hexafluorophosphate), HOBt (hydroxybenzotriazole), N,Nꢀ
diisopropylethylamine (DIEA), TIS (triisopropylsilane).
The UVꢀvis absorption spectra of TPEꢀN3 and AcꢀDEVDKꢀTPE in DMSO/water (v/v = 1/199) were
shown in Figure S9A in the SI. Both have a similar absorption profile with an obvious absorbance in the
300−350 nm range. It is known that AIE fluorogen is virtually nonꢀfluorescent in good solvents but
emits intensely when aggregated in poor solvents. As can be seen from the photoluminescence (PL)
spectra shown in Figure 1A, TPEꢀN3, a hydrophobic AIE fluorogen, shows intense fluorescence as
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nanoaggregates in a mixture of DMSO/water (v/v = 1/199) with a quantum yield (Φ) of 0.2, while the
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AcꢀDEVDKꢀTPE probe is almost nonꢀfluorescent in the same medium (Φ = 0.001), due to its good
solubility in water. The aggregate formation of the former and the molecular dissolution of the latter
were confirmed by laser light scattering (LLS) measurements. In the aqueous mixture, the hydrophobic
TPEꢀN3 molecules cluster into aggregates with an average diameter of 126 nm (SI Figure S9B). No LLS
signals, however, could be detected from the solution of AcꢀDEVDKꢀTPE.
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casp-3
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TPE-N3
Ac-DEVDK-TPE
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Figure 1. (A) Photoluminescence (PL) spectra of TPEꢀN3 and AcꢀDEVDKꢀTPE in DMSO/water (v/v =
1:199). Inset: the photographs of TPEꢀN3 and AcꢀDEVDKꢀTPE in DMSO/water (v/v = 1:199) taken
under illumination of a UV lamp. (B) PL spectra of AcꢀDEVDKꢀTPE upon treatment with caspaseꢀ3
and caspaseꢀ7 in the presence and absence of inhibitor 5ꢀ[(S)ꢀ(+)ꢀ2ꢀ(methoxymethyl)pyrrolidino]
sulfonylisatin. (C) PL spectra of AcꢀDEVDKꢀTPE in the presence of different amounts of caspaseꢀ3 (0,
7, 35, 50, 70, 100 and 200 pM). (D) PL spectra of different concentrations of AcꢀDEVDKꢀTPE (0, 1, 2,
5, 10 and 20 ꢁM) in the presence of caspaseꢀ3. The incubation time is 1 h. [TPEꢀN3] = [AcꢀDEVDKꢀ
TPE] = 10 ꢁM; [caspaseꢀ3] = [caspaseꢀ7] = 100 pM; [inhibitor] =10 ꢂM; λex = 312 nm.
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As biosensing is often conducted in buffers, it is important to study the effect of ionic strength on the
emission behavior of the probe. The experiments were performed with addition of sodium chloride into
an aqueous solution of AcꢀDEVDKꢀTPE (10 ꢁM). Almost no change in the PL spectrum of the probe is
observed when the concentration of NaCl is increased from 0 to 960 mM (SI Figure S10). Clearly, ionic
strength does not affect the fluorescence property of AcꢀDEVDKꢀTPE. Its PL profile also does not
change in the presence of the Dulbecco’s Modified Eagle Medium (DMEM), which contains amino
acids, salts, glucose and vitamins. The probe maintains an “off” state in the complex environment and
thus has great potential to serve as a specific lightꢀup probe with minimum background interference.
Ac-DEVDK-TPE Probe for Detection of Caspase Activities in Solutions. The distinct emission
behavior of AcꢀDEVDKꢀTPE as compared to TPE itself prompts us to explore the potential of the probe
for caspase activity study. We performed in vitro enzymatic assays with recombinant caspaseꢀ3 and
caspaseꢀ7 first. Mixtures of AcꢀDEVDKꢀTPE (10 ꢁM) and caspases (100 pM) were prepared and
incubated in piperazineꢀN,N’ꢀbis(2ꢀethanesulfonic acid) (PIPES) buffer (50 mM PIPES, 100 mM NaCl,
1
mM ethylenediaminetetraacetic acid, 0.1% w/v 3ꢀ[(3ꢀcholamidopropyl)dimethylammonio]
o
propanesulfonic, 25% w/v sucrose, pH = 7.2) at 37 C. After 1 h incubation, the PL spectra were
measured in the range from 360 to 620 nm. As shown in Figure 1B, strong fluorescence signals are
recorded for the probe upon treatment with caspaseꢀ3 and caspaseꢀ7. However, most of the fluorescence
is readily competed away by pretreatment of the probe with 5ꢀ[(S)ꢀ(+)ꢀ2ꢀ(methoxymethyl)pyrrolidino]
sulfonylisatin, a highly specific inhibitor of caspaseꢀ3/7,35 indicating that specific cleavage of DEVD
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from AcꢀDEVDKꢀTPE is inhibited. This is further confirmed by LCꢀMS as shown in Figure S11 in the
SI. After treatment with caspaseꢀ3, particles with an average diameter of 133 nm are formed along with
the increase of solution fluorescence (SI Figure S12A).
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To optimize the quantity of enzyme and probe used in the assays, different concentrations of caspaseꢀ
3 ranging from 0 to 200 pM were incubated with AcꢀDEVDKꢀTPE (10 ꢂM) in PIPES buffer (pH = 7.2)
for 1 h. Figure 1C shows the variation in the PL spectra of the assay. With the increasing concentrations
of caspaseꢀ3, the PL intensities gradually increase due to the increased amount of TPEꢀK fluorogen (Φ
= 0.07, SI Table S1) released which emits strongly in aqueous solution. In comparison to the probe’s
intrinsic emission in the buffer solution, a 75ꢀfold PL enhancement is observed when the probe is
incubated with 200 pM caspaseꢀ3 for 1 h. Additionally, the PL intensities of the assay increase linearly
with the increasing concentrations of caspaseꢀ3 (SI Figure S12B). Therefore, the hydrolysis kinetics of
AcꢀDEVDKꢀTPE catalyzed by caspaseꢀ3 can be easily studied based on the PL intensity changes.
Likewise, when recombinant caspaseꢀ3 (100 pM) is treated with different concentrations of Acꢀ
DEVDKꢀTPE, a progressive fluorescence increase at 470 nm is also observed with increasing
concentrations of AcꢀDEVDKꢀTPE. As there is only slight fluorescence increase observed when the
concentration of AcꢀDEVDKꢀTPE is higher than 10 ꢁM (Figure 1D), these data illustrate that 10 ꢂM
AcꢀDEVDKꢀTPE is sufficient for digestion by 100 pM caspaseꢀ3. Therefore, 10 ꢂΜ AcꢀDEVDKꢀTPE
and 100 pM caspaseꢀ3 were chosen as the optimal conditions for the following enzymatic experiments.
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The enzyme kinetic studies by incubating recombinant caspaseꢀ3/7 with AcꢀDEVDKꢀTPE in buffer
at 37 °C were subsequently performed, and the changes in fluorescence were monitored over time. As
shown in Figure 2A, a significant increase in solution fluorescence over background is observed when
caspaseꢀ3/7 is used. In the absence of caspaseꢀ3/7, no change in fluorescence is observed, confirming
that AcꢀDEVDKꢀTPE is specifically recognized and cleaved by caspaseꢀ3/7. To further investigate the
probe selectivity, AcꢀDEVDKꢀTPE was treated with several proteins, such as caspaseꢀ3/7, pepsin, BSA,
trypsin, and lysozyme, under identical conditions. As shown in Figure 2B, caspaseꢀ3/7 display around
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45 and 28ꢀfold higher changes in (I−I0)/I0 than the other four proteins. This substantiates that Acꢀ
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Figure 2. (A) Timeꢀdependent PL spectra of AcꢀDEVDKꢀTPE upon addition of caspaseꢀ3 and caspaseꢀ
7 from 0 to 120 min. [caspaseꢀ3] = [caspaseꢀ7] = 100 pM. (B) Plot of (I I0)/I0 versus different proteins,
−
where I and I0 are the PL intensities at protein concentrations of 100 and 0 pM, respectively. Inset:
photographs of the corresponding solutions containing caspaseꢀ3 or pepsin taken under illumination of a
UV lamp. The solutions containing other proteins (lysozyme, trypsin and BSA) look the same as that of
pepsin. (C) Plot of (I−I0)/I0 versus concentrations of AcꢀDEVDKꢀTPE, AcꢀDEVDꢀAFC and ZꢀDEVDꢀ
AFC in PIPES buffer, where I and I0 are the PL intensities of mixtures with or without substrates. (D)
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Timeꢀdependent PL spectra of AcꢀDEVDKꢀTPE in apoptotic MCFꢀ7 cell lysate (ApꢀMCFꢀ7, black
squares) and normal MCFꢀ7 cell lysate (NorꢀMCFꢀ7, red circles). [AcꢀDEVDKꢀTPE] = 10 ꢁM; λex =
312 nm.
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Kinetic Analysis of Ac-DEVDK-TPE Cleavage. We next performed the kinetic analysis of Acꢀ
DEVDKꢀTPE cleavage by caspaseꢀ3. Caspaseꢀ3 (100 pM) was incubated at 37 °C in 50 ꢂL of PIPES
buffer with increasing concentrations of AcꢀDEVDKꢀTPE and two commercial coumarinꢀbased
caspaseꢀ3 substrates, AcꢀDEVDꢀAFC and ZꢀDEVDꢀAFC (from 0 to 100 ꢂM) in parallel. The
fluorescence intensities at 470 or 500 nm were monitored over 60 min at 37 °C. Kinetic constants were
computed by direct fitting the data to the MichaelisꢀMenton Equation V0 = Kcat [E]0[S]/Km + [S] using a
nonꢀlinear regression via GraphPad Prism software.36 The Michaelis constant Km is a measure of the
substrate’s affinity to the enzyme and it is commonly used to evaluate the effect of modification on
substrate. Kcat, the turnover number, is the maximum number of substrate molecules converted to
product per enzyme molecule per second. The Km and Kcat values of AcꢀDEVDKꢀTPE against caspaseꢀ3
are 5.38 ± 0.03 ꢂΜ and 17.1 ± 0.2 sꢀ1 respectively, which are apparently better than those of AcꢀDEVDꢀ
AFC (Km = 12.70 ± 0.02 ꢂΜ and Kcat = 2.7 ± 0.1 sꢀ1) and ZꢀDEVDꢀAFC (Km = 15.37 ± 0.01 ꢂΜ and
Kcat = 2.5 ± 0.3 sꢀ1) (Figure 2C, SI Figure S13 and Table S2). The higher binding affinity to caspaseꢀ3
and larger enzyme turnover number for AcꢀDEVDKꢀTPE as compared to those for AcꢀDEVDꢀAFC and
ZꢀDEVDꢀAFC clearly indicate the great potentials of the developed probe for enzyme activity studies.
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Owing to the essential roles caspaseꢀ3/7 played in cellular apoptosis, we next assessed whether Acꢀ
DEVDKꢀTPE could selectively monitor the caspaseꢀ3/7 activity in complex cellular proteomes. Both
normal and apoptotic MCFꢀ7 cellular lysates were collected and directly treated with AcꢀDEVDKꢀTPE
followed by fluorescence measurement in a timeꢀdependent manner. No fluorescence increase is
observed in the unꢀinduced cellular lysates, while a typical saturation kinetic of enzymatic activity is
observed for apoptotic lysates (Figure 2D). This is in consistent with recombinant caspaseꢀ3/7 shown in
Figure 2A, which further demonstrates that the fluorescence is generated from catalytic cleavage of the
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substrate DEVD in AcꢀDEVDKꢀTPE.
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To explore the possibility of using AcꢀDEVDKꢀTPE as a screening tool for caspaseꢀ3 inhibitors, we
performed the competitive enzymatic assay with different amounts of 5ꢀ[(S)ꢀ(+)ꢀ2ꢀ
(methoxymethyl)pyrrolidino]sulfonylisatin (SI Figure S14). Results show a dose dependent decrease of
fluorescence with a calculated IC50 value of 63.4 nM for the inhibitor, which is in good agreement with
the literature value reported previously.35 These results indicate that our probe has great capability for
caspase inhibitor screening.
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Anti Casp-3
Overlay
Figure 3. Fluorescence microscope images. (A−C) Normal MCFꢀ7 cells treated with AcꢀDEVDKꢀTPE;
(D−F) Apoptotic MCFꢀ7 cells treated with AcꢀDEVDKꢀTPE (5 ꢂM, 1% DMSO); (G−I) Apoptotic
MCFꢀ7 cells treated with AcꢀDEVDKꢀTPE (5 ꢂM, 1% DMSO), inhibitor (10 ꢂM) and caspaseꢀ3
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antibody. Staurosporine (STS, 1 ꢁM) was used to induce cell apoptosis. Blue = probe fluorescence; red
= immunofluorescence signal generated from antiꢀcaspaseꢀ3 primary antibody and a Texas Red labeled
secondary antibody. The images were acquired using fluorescence microscope (Nikon) equipped with
DAPI and Texas Red. All images share the same scale bar (10 ꢂm).
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Imaging of Cell Apoptosis Using Ac-DEVDK-TPE. After investigating the response characteristics
of AcꢀDEVDKꢀTPE as a protease probe in vitro, we further explored the potential of the probe for liveꢀ
cell imaging of caspaseꢀ3 activation. Cytotoxicity of AcꢀDEVDKꢀTPE was first evaluated by the widely
used MTT assay. As shown in Figure S15 in the SI, after being incubated with 5, 10, or 25 ꢁM Acꢀ
DEVDKꢀTPE for 48 h, the cell viabilities are close to 100% under the testing conditions, indicative of
low cytotoxicity of the probe. Fluorescence microscopy was used to image normal and apoptotic MCFꢀ7
cells after treatment with AcꢀDEVDKꢀTPE. MCFꢀ7 cells were first incubated with AcꢀDEVDKꢀTPE in
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DMEM for 2 h at 37 C. The cells were subsequently treated with staurosporine (STS, 1 ꢁM), a
commonly used apoptosis inducer. After 1 h incubation, AcꢀDEVDKꢀTPE activation was determined by
monitoring the fluorescence changes with a fluorescence microscopy. As shown in Figure 3 and Figure
S16 in the SI, normal, unꢀinduced cells show an extremely low fluorescence signal, indicative of little or
no caspaseꢀ3 activity (Figure 3A). In sharp contrast, strong fluorescence signals are collected from the
cells treated by STS (Figure 3D). The signals are greatly reduced when STSꢀinduced cells are preꢀ
treated with 5ꢀ[(S)ꢀ(+)ꢀ2ꢀ(methoxymethyl)pyrrolidino]sulfonylisatin, a commercial caspaseꢀ3/ꢀ7
inhibitor, before incubation with AcꢀDEVDKꢀTPE (Figure 3G). Furthermore, excellent overlap is
observed between the fluorescence images of the probe and immunofluorescence signals generated from
antiꢀcaspaseꢀ3 primary antibody and a Texas Red labeled secondary antibody (Figure 3F). Additionally,
apoptotic MCFꢀ7 cells were treated with both AcꢀDEVDKꢀTPE and commercial Annexin VꢀAlexa
Fluor. As expected, Annexin VꢀAlexa Fluor is localized on the cell surface, but AcꢀDEVDKꢀTPE shows
strong fluorescence inside the cells (SI Figure S17). Collectively, these results provide direct evidence
for intracellular delivery and caspaseꢀspecific activation of the imaging probe. Undoubtedly, Acꢀ
DEVDKꢀTPE is a suitable probe for detection of caspaseꢀ3 activity and apoptosis imaging in live cells.
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15 min 30 min
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Figure 4. Realꢀtime fluorescence images showing the cell apoptotic process of MCFꢀ7 cells with Acꢀ
DEVDKꢀTPE at room temperature. STS (1 ꢂM) was used to induce cell apoptosis. The images were
acquired using fluorescence microscope (Nikon) equipped with DAPI filter. All images have the same
scale bar (20 ꢂm). See the movie in the SI for the dynamic imaging process.
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To explore whether our probe can be used for monitoring cell apoptosis, realꢀtime imaging
o
experiments were performed. AcꢀDEVDKꢀTPE (5 ꢂM) was first incubated with MCFꢀ7 cells at 37 C.
After 2 h incubation, the cells were treated with STS (1 ꢂM) and monitored with fluorescence
microscopy to obtain realꢀtime fluorescence images. The dark background in each image shown in
Figure 4 indicates that the probe is nonꢀfluorescent in the cell culture media. As the incubation time
elapses, the fluorescence intensity increases gradually with the cellular apoptotic progress, which
reaches a maximum at 90 min. These results clearly demonstrate that AcꢀDEVDKꢀTPE not only can be
used for detection of caspaseꢀ3 activity but also has the potential for realꢀtime monitoring of cell
apoptosis.
In Situ Screening of Apoptosis-inducing Agents. The discovery of novel compounds that modulate
apoptosis pathways could lead to the development of new anticancer agents. To assess the capability of
our probe for in situ screening of compounds that can induce cell apoptosis, three known apoptosis
inducers, sodium ascorbate,37 cisplatin and STS,38 were used to treat MCFꢀ7 cells. After the cells were
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incubated with AcꢀDEVDKꢀTPE for 2 h, each compound (1 ꢂM in DMSO) was added into the chamber
for additional 1.5 h incubation. The apoptosisꢀinducing capabilities of these agents were evaluated by
monitoring the cell fluorescence increase with a fluorescence microscopy. As shown in Figure 5A, a 35ꢀ
fold fluorescence increase is observed for STSꢀtreated cells compared to that for DMSOꢀtreated cells,
while only 11 and 12ꢀfolds are observed for sodium ascorbate and cisplatin treated cells, respectively.
These results indicate that STS has a relatively high inducing efficacy for apoptosis, which is consistent
with the literature report.38 Therefore, our probe can potentially be used for screening apoptosisꢀ
inducing agents in living cells.
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To further evaluate whether the probe can be used to quantify the efficacy of apoptosis inducing
agents, after incubation with AcꢀDEVDKꢀTPE, MCFꢀ7 cells were treated with different amounts of
compounds, namely DMSO, sodium ascorbate, cisplatin and STS for 2 h before the fluorescence
measurement. As shown in Figure 5B, with increasing concentration of the compounds used, the cell
fluorescence is progressively intensified. Among them, STS shows the best performance for apoptosis
induction. Collectively, these results suggest that our probe can also be used for quantitative analysis of
apoptosisꢀrelated drug efficacy in living cells.
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treatment with 1 ꢂM each of DMSO, sodium ascorbate (Na asb), cisplatin, and staurosporine (STS).
[AcꢀDEVDKꢀTPE] = 5 ꢁM. Nuclei were stained with propidium iodide (Invitrogen). The images were
acquired using fluorescence microscope (Nikon) equipped with DAPI and Texas Red filter. All images
share the same scale bar (10 ꢂm). (B) Photoluminescence intensities of AcꢀDEVDKꢀTPE preꢀincubated
MCFꢀ7 cells upon treatment with different amounts of DMSO, Na asb, cisplatin, STS. λex = 312 nm; λem
= 470 nm.
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CONCLUSIONS
In summary, a DEVDꢀconjugated AIE probe has been successfully developed in this work. Thanks
to its novel AIE nature, the probe is nonfluorescent in aqueous buffers but becomes emissive when
cleaved by caspaseꢀ3/ꢀ7. It enables lightꢀup monitoring of caspaseꢀ3/ꢀ7 activities in solution and in cells
with high signal to noise ratios, which shows superior performance over commercial coumarin based
fluorogenic probes. Additionally, our AIE probe strategy provides an efficient platform for realꢀtime
imaging of live cell apoptosis, which further allows in situ screening and quantification of apoptosisꢀ
inducing agents. In light of its simplicity, lowꢀcost and high efficiency as a live cell apoptosis imaging
probe, further tuning of the emission spectrum of AIE fluorogen to red and nearꢀIR region will facilitate
the development of specific bioprobes for in vivo imaging of cell apoptosis and drug screening.
EXPERIMENTAL SECTION
General Information. Bovine serum albumin (BSA), lysozyme, pepsin, and trypsin were purchased from
Sigma. Recombinant human caspaseꢀ3, caspaseꢀ7, AcꢀDEVDꢀAFC were purchased from R&D Systems. Zꢀ
DEVDꢀAFC and Annexin VꢀAlexa Fluor were purchased from Invitrogen. Inhibitor 5ꢀ[(S)ꢀ(+)ꢀ2ꢀ
(methoxymethyl)pyrrolidino]sulfonylisatin was purchased from Calbiochem. Cleaved Caspaseꢀ3 (Asp175)
(5A1E) Rabbit mAb (#9664) was purchased from Cell Signaling. Mouse antiꢀrabbit IgGꢀTR (scꢀ3917) was
purchased from Santa Cruz. Fetal bovine serum (FBS) and trypsinꢀEDTA solution were purchased from Gibco
(Lige Technologies, AG, Switzerland). Staurosporine was purchased from Biovision. MilliꢀQ water was supplied
by MilliꢀQ Plus System (Millipore Corporation, Breford, USA). MCFꢀ7 breast cancer cell line was provided by
American Type Culture Collection. UVꢀvis absorption spectra were taken on a Milton Ray Spectronic 3000 array
spectrophotometer. Photoluminescence (PL) spectra were measured on a PerkinꢀElmer LS 55 spectrofluorometer.
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All PL spectra were measured with an excitation wavelength of 312 nm. Average particle size and size
distribution of TPEꢀN3 and lysineꢀconjugated TPE (KꢀTPE) were determined by laser light scattering (LLS) with
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particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90 at room temperature.
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The cells were imaged by fluorescence microscope (Nikon A1 Confocal microscope).
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Hexane and tetrahydrofuran (THF) were distilled from sodium benzophenoneketyl immediately prior to use.
Dichloromethane (DCM) was distilled over calcium hydride. Diphenylmethane, nꢀbutyllithium, 4ꢀ
methylbenzophenone, pꢀtoluenesulfonic acid, Nꢀbromosuccinimide, benzoyl peroxide, copper(II) sulfate, sodium
ascorbate, N,Nꢀdiisopropylethylamine (DIEA), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA),
triisopropylsilane (TIS), piperazineꢀN,N′ꢀbis(2ꢀethanesulfonic acid (PIPES), ethylenediaminetetraacetic acid
(EDTA), 3ꢀ[(3ꢀcholamidopropyl)dimethylammonio]propanesulfonic acid (CHAPS), hexꢀ5ꢀynoic acid and
solvents were all purchased from SigmaꢀAldrich and used as received without further purification. Rinkꢀamide
resin, OꢀbenzotriazoleꢀN,N,N’,N’ꢀtetramethylꢀuroniumꢀhexafluoroꢀphosphate (HBTU), Nꢀhydroxybenzotriazole
(HOBt) and Fmocꢀprotective amino acids were purchased from GL Biochem Ltd. 1H and 13C 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 (TMS)
Si(CH3)4 = 0 ppm). Highꢀresolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass
Spectrometer System operating in a MALDIꢀTOF mode. The HPLC profiles and ESI mass spectra were acquired
using a Shimadzu ITꢀTOF. A 0.1% TFA/H2O and 0.1% TFA/acetonitrile were used as eluents for all HPLC
experiments. The flow rate was 0.6 mL/min for analytical HPLC and 3 mL/min for preparative HPLC. Details of
synthetic procedures and characterizations of compounds (1, 2 and 5) are reported in the Supporting Information.
Characterizations of the key intermediates and AcꢀDEVDKꢀTPE are reported below.
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Synthesis of 1-((4-azidomethyl)phenyl)-1,2,2-triphenylethene (3): In a 250 mL twoꢀneck round bottom flask,
1.70 g (4 mmol) of 2 and 0.39 g (6 mmol) of sodium azide were dissolved in DMSO under N2. The mixture was
stirred at room temperature overnight. A large amount (100 mL) of water was then added and the solution was
extracted three times with diethyl ether. The organic layers were combined, dried over magnesium sulfate and
concentrated. The crude product was purified by silicaꢀgel chromatography using hexane/chloroform (v/v = 1:9)
as eluent to give 3 as a white solid (1.5 g, 97% yield). 1H NMR (CDCl3, 400 MHz), δ (TMS, ppm): 7.13−7.06 (m,
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9H), 7.06−6.98 (m, 10H), 4.24 (s, 2H). C NMR (CDCl3, 100 MHz), δ (TMS, ppm): 143.27, 142.90, 142.83,
140.82, 139.62, 132.61, 131.22, 131.11, 130.67, 127.09, 127.04, 126.99, 126.02, 125.90, 53.91. HRꢀMS
(MALDIꢀTOF): m/z 387.1342 [(M)+, calcd. 387.1735].
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Synthesis, Purification and Characterization of Ac-DEVDK-alkyne (Ac-DEVDK-A). Ac-DEVDKꢀA was
synthesized using standard Fmoc strategy with rink amide resin as the solid support. Standard
HOBt/HBTU/DIEA coupling method was used throughout the whole process.18d The resin (100 mg, loading ∼0.5
mmol/g) was swelled in HPLCꢀgrade DMF for 1 h at room temperature. Subsequently, Fmoc group was
deprotected in piperidine/DMF (v/v = 1/4) for 2 h at room temperature. Following piperidine removal, the resin
was washed extensively with DMF and DCM and dried thoroughly under high vacuum. Next, alkyneꢀcontaining
lysine 5 (SI Scheme S1) was dissolved in dry DMF (1.5 mL) together with HBTU (4 equiv), HOBt (4 equiv), and
DIEA (8 equiv). The dry resin was then added and the resulting mixture was shaken at room temperature. After
overnight reaction, the resin was filtered and washed thoroughly with DMF (3×), DCM (3×) and DMF (3×) until
the filtrate became colorless. After drying thoroughly under high vacuum, the resin was deprotected again with
20% piperidine in DMF for the next coupling cycle. The above cycle was repeated until the last amino acid has
been coupled. Finally, the resin was capped with a solution of Ac2O (10 eq) and DIEA (20 eq) in DCM (200 mL),
and the mixture was allowed to react for 2 h at room temperature. After the whole coupling process, the resin was
washed thoroughly with DMF and dried under high vacuum for 2 h at room temperature. The peptide was then
cleaved in a mixture of 95% TFA, 2.5% triisopropylsilane (TIS) and 2.5% H2O for 4 h at room temperature.
Following prolonged concentration in vacuum until >80% of cleavage cocktail was removed, cold ether (chilled
to −20 °C) was added to the liquid residue to precipitate the peptide. The ether layer was then decanted and the
precipitates were dried thoroughly in vacuum. The resulting peptide was further purified by prepꢀHPLC and
characterized by LCꢀMS. ITꢀTOF m/z calcd: 739.34, found 739.30. The HPLC condition is: 20−100% B for 10
min, then 100% B for 2 min, 20% B for 5 min (Solvent A: 100% H2O with 0.1% TFA; Solvent B: 100% CH3CN
with 0.1% TFA).
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“Click” Synthesis of Probe Ac-DEVDK-TPE. AcꢀDEVDꢀA (3.7 mg, 5 ꢁmol) and TPEꢀN3 (2.2 mg, 6 ꢁmol)
were dissolved in 50 ꢁL of DMSO. A mixture of DMSO/H2O solution (v/v = 1/1; 0.5 mL) was subsequently
added and the reaction was shaken for a few minutes to obtain a clear solution. The “click” reaction was initiated
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by sequential addition of catalytic amounts of sodium ascorbate (0.4 mg, 2.0 ꢁmol) and CuSO4 (1.6 mg, 1.0
ꢁmol). The reaction was continued with shaking at room temperature for another 24 h. The final product was
purified by prepꢀHPLC and further characterized by NMR and HRMS. 1H NMR (400 MHz, DMSOꢀd6), δ (TMS,
ppm): 8.25 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.73ꢀ7.67 (m, 3H),
7.12ꢀ6.95 (m, 21H), 5.42 (s, 2H), 4.55ꢀ4.50 (m, 2H), 4.29ꢀ4.24 (m, 1H), 4.12ꢀ4.04 (m, 2H), 2.98ꢀ2.94 (m, 2H),
2.74ꢀ2.70 (dd, J = 8.0 Hz, 1H), 2.65ꢀ2.60 (dd, J = 8.0 Hz, 1H), 2.57ꢀ2.42 (m, 5H, overlap with d6ꢀDMSO), 2.23ꢀ
2.17 (m, 2H), 2.08 (t, J = 8.0 Hz, 2H), 1.93ꢀ1.89 (m, 2H), 1.82 (s, 3H), 1.77 (t, J = 8.0 Hz, 2H), 1.70ꢀ1.61 (m,
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1H), 1.50ꢀ1.40 (m, 1H), 1.40ꢀ1.27 (m, 2H), 1.26ꢀ1.18 (m, 2H), 0.80 (m, 6H). 13C NMR (100 MHz, DMSOꢀd6),
δ
(TMS, ppm): 174.9, 174.3, 172.9, 172.6, 172.4, 171.9, 171.7, 171.6, 171.0, 170.4, 147.6, 143.9, 143.8, 141.8,
140.9, 135.1, 131.7, 131.4, 128.7, 128.1, 127.5, 127.4, 123.0, 58.4, 55.3, 53.1, 52.9, 50.4, 50.3, 39.3, 36.8, 36.5,
35.7, 32.3, 31.5, 30.9, 29.7, 27.9, 26.0, 25.5, 23.5, 23.3, 20.0, 18.8. HRMS (MALDIꢀTOF): m/z 1149.4998
([M+Na]+, calcd 1149.5124). The HPLC condition is: 20ꢀ100% B for 10 min, then 100% B for 2 min, 20% B for
5 min (Solvent A: 100% H2O with 0.1% TFA; Solvent B: 100% CH3CN with 0.1% TFA).
General Procedure for Enzymatic Assay. DMSO stock solutions of AcꢀDEVDKꢀTPE were diluted with
caspaseꢀ3/7 assay buffer (50 mM PIPES, 100 mM NaCl, 1 mM EDTA, 0.1% w/v CHAPS, 25% w/v sucrose, pH
= 7.2) to make 10 ꢂM working solutions. 5 ꢁL of the recombinant caspaseꢀ3 and ꢀ7 (∼0.04 ꢂg/ꢂL stock solution
in assay buffer) was added into the above working solution. The reaction mixture was incubated at room
temperature for 60 min and was then diluted to a total of 300 ꢁL with deionized water for photoluminescence
measurement. The solution was excited at 312 nm, and the emission was collected from 360 to 620 nm.
Cell Culture. MCFꢀ7 cell lines were provided by American Type Culture Collection. MCFꢀ7 breast cancer
cells were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% heatꢀinactivated fetal bovine serum
(FBS; Invitrogen), 100 U/mL penicillin and 100 ꢁg/mL streptomycin (Thermo Scientific) and maintained in a
humidified incubator at 37 °C with 5% CO2. Before experiment, the cells were preꢀcultured until confluence was
reached.
Microscopy Imaging. MCFꢀ7 cells were cultured in the chambers (LABꢀTEK, Chambered Coverglass
System) at 37 oC. After 80% confluence, the adherent cells were washed twice with 1× PBS buffer. The DEVDKꢀ
o
TPE solution (5 ꢁM, 0.3 mL) was then added to the chamber. After incubation for 2 h at 37 C, the cells were
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washed once with 1× PBS buffer, and treated with 1 ꢂM apoptosis inducers (staurosporine, sodium ascorbate and
cisplatin) for 1 h. The cells were washed one time with 1× PBS buffer. For coꢀlocalization with Annexin VꢀAlexa
Fluor, the cells were further incubated with a mixture of Annexin VꢀAlexa Fluor/FBSꢀfree DMEM (1:799 by vol)
for 15 min at room temperature, washed once with 1× PBS buffer. The cells were then kept in fresh FBSꢀfree
DMEM for cell imaging. For coꢀlocalization with active caspaseꢀ3 antibody, the cells were first fixed for 15 min
with 3.7% formaldehyde in 1× PBS at room temperature, washed twice with cold 1× PBS again, and
permeabilized with 0.1% Triton Xꢀ100 in 1× PBS for 10 min. The cells were then blocked with 2% BSA in 1×
PBS for 30 min, washed twice with 1× PBS. The cells were subsequently incubated with a mixture of antiꢀ
caspaseꢀ3 antibody/1× PBS (1:99 by vol) for 1 h at room temperature, washed once with 1× PBS buffer and then
incubated with Mouse antiꢀrabbit IgGꢀTR (0.8 ꢁg mLꢀ1) in 1× PBS for 1 h, following by washing with 1× PBS
again. The imaging was done with fluorescence microscope (Nikon) equipped with DAPI, FITC and Texas Red
filters.
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Real-time Imaging of Cell Apoptosis. MCFꢀ7 cells were cultured in the 8ꢀwell chambers (LABꢀTEK,
o
Chambered Coverglass System) at 37 C. After 80% confluence, the adherent cells were washed twice with 1×
PBS buffer. The DEVDKꢀTPE solution (5 ꢁM, 0.3 mL) was then added to the chamber. After 2 h incubation at
o
37 C, the cells were washed twice with 1× PBS buffer, and staurosporine (1 ꢁM, 0.3 mL) was added to the
chambers. The chambers were then placed on the microscope platform immediately and the microscope focused
on a collection of cells. The fluorescence images at DAPI channel were acquired every 2 min.
Quantification of Cell Apoptosis by Fluorescence Microplate Reader. MCFꢀ7 cells were seeded in 96ꢀwell
plates (Costar, IL, USA) at an intensity of 4 × 104 cells mLꢀ1. After 80% confluence, the medium was replaced by
o
10 ꢂM AcꢀDEVDKꢀTPE in FBS free DMEM medium. After 2 h incubation at 37 C, the adherent cells were
washed twice with 1× PBS buffer, and 100 ꢂL different concentrations of STS in DMEM were added into the
o
wells. After additional 2 h incubation at 37 C, the cells were washed once with 1× PBS buffer followed by
fluorescence measurement using TꢀCAN microplate reader. The excitation and emission wavelengths are 312 and
470 nm, respectively.
ACKNOWLEDGMENTS. We thank the Singapore National Research Foundation (Rꢀ279ꢀ000ꢀ323ꢀ
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281), the Temasek Defense Systems Institute (R279ꢀ000ꢀ305ꢀ232/422/592), the Research Grants
Council of Hong Kong (HKUST2/CRF/10and N_HKUST620/11), and the Institute of Materials
Research and Engineering of Singapore (IMRE/11ꢀ1C0213) for financial support.
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Supporting Information Available. Experimental procedures for intermediates; structural
characterization data of TPEꢀN3 and AcꢀDEVDKꢀTPE; particle size distribution of TPEꢀN3 and KꢀTPE;
absorption spectra of TPEꢀN3 and AcꢀDEVDKꢀTPE; movie showing the dynamic process of cell
apoptosis imaging. This material is available free of charge via the Internet at http://pubs.acs.org.
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SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”).
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Apoptotic Cell
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Caspꢁ3/7
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Internalization
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DEVDKꢁTPE
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