A cascade enzymatic reaction activatable gemcitabine prodrug with an AIE-based intracellular light-up apoptotic probe for: In situ self-therapeutic monitoring

Article abstract of DOI:10.1039/c7cc04872f

A targeted cathepsin B-activatable gemcitabine prodrug with caspase-3 specific light-up tetraphenylene (TPE) as an apoptotic probe based on aggregation-induced emission (AIE) properties was designed for in situ self-therapeutic monitoring of pancreatic ca

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Cascade enzymatic reaction activatable gemcitabine prodrug with
AIE-based intracellular light-up apoptotic probe for in situ self-
therapeutic monitoring
Received 00th January 20xx,
Accepted 00th January 20xx
Haijie Han,a Wenzhuo Teng,a Tingting Chen,a Jue Zhao,b Qiao Jin,*a,c Zhihui Qin,a and Jian Ji*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
A targeted cathepsin B-activatable gemcitabine prodrug with Though magnetic resonance imaging (MRI) is widely used in
caspase-3 specific light-up tetraphenylene (TPE) as apoptotic estimating the effectiveness of pancreatic cancer treatment by
probe based on aggregation-induced emission (AIE) properties measuring the tumour size, it still has the problem in
was designed for in situ self-therapeutic monitoring of pancreatic evaluating the early response of cancer to specific treatment
cancer cells.
as the change in tumour size is not obvious at the early stage
of therapy. One of the most promising strategies is to combine
an apoptotic probe into the system.9 When the activated GEM
kills pancreatic cancer cells by inducing cell apoptosis, the
apoptotic probe can be used to evaluate the therapeutic effect
simultaneously. As is known, caspase-3 plays an important role
in signal transforming in the procedure of cell apoptosis. It was
already reported that caspase-3 could be activated by
chemotherapeutic agents including GEM.10
With a 5-year survival rate of only 5% and a median survival of
6 months, pancreatic cancer is a notoriously lethal cancer
worldwide.1 Owing to a lack of effective diagnostic methods,
more than 80% of patients with unresectable locally advanced
or metastatic pancreatic cancer are restricted to palliative
treatment and gemcitabine (GEM) has been the standard first-
line drug for these patients since 1997.2 However, the clinical
application of GEM is severely limited due to the rapid
deamination of GEM with an extremely short plasma
circulation time.3 Our group and others have found the use of
GEM prodrugs could effectively avoid the deactivation of
GEM.4
Up to date, fluorescent probes with aggregation-induced
emission (AIE) characteristics have been widely reported and
utilized as chemosensors and fluorescent bioprobes.11
Tetraphenylethene (TPE), for instance, shows weak or no
fluorescence in the molecularly dissolved state but strong blue
fluorescence in the aggregated state on account of the
restriction of intramolecular rotations (RIR), which can be
utilized for cellular imaging and medical imaging.12 TPE has
many advantages in biomedical applications, such as easy
synthesis and functionalization, strong and stable fluorescence,
and low cytotoxicity. Due to these unique characteristics, it is
of particular interest in developing AIE-based stimuli-
responsive light-up probe for specific biological processes. As
is known, among the endogenous stimuli, such as pH, redox
potential and hypoxia in pathological conditions, the
dysregulation of certain enzymes is a more specific character
for many types of tumours.13 For instance, lysosomal protease
cathepsin B, overexpressed in many types of malignant
tumours, was widely exploited for enzyme-responsive drug
delivery.14
An early assessment of therapeutic efficiency on pancreatic
cancer is particularly important in clinic since it can minimize
the possible ineffective therapy duration.5 GEM, a non-
fluorescent drug, can be hardly detected in the process of GEM
delivery or release.4a Recently, some theranostic GEM delivery
systems loaded with fluorescent dyes were reported for real-
time monitoring of GEM delivery.6 Furthermore, strategies for
real-time monitoring of GEM release via GEM release-induced
fluorescence intensity change upon tumour-associated stimuli
were developed.7 However, in these works, in situ evaluation
of therapeutic effect could hardly be realized, which means
extra detections are required for therapeutic evaluation.
Herein, a targeted theranostic GEM (prodrug 1, Scheme 1)
with cancer cell overexpressed cathepsin B-sensitive GEM
release and subsequent caspase-3 triggered AIE-based
intracellular light-up was developed as an apoptotic probe for
therapeutic monitoring of pancreatic cancer cells. This prodrug
consisting of RGD peptide as the targeting moiety is expected
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to be accumulated preferentially in pancreatic cancer cells almost no GEM release from prodrug 1 in the absence of
with overexpressed αvβ3 integrin.15 Following cellular cathepsin B. In addition, only few GEM was released from
internalization, the GFLG peptide in GEM prodrug is prodrug 2 with stable GAAG peptide sequence in the presence
hydrolysed in the presence of overexpressed cathepsin B,4b, 14a, of cathepsin B. These results suggested that prodrug
16
1 could
followed by the release of the active drug (GEM) and the generate pharmaceutically active GEM trigged by cathepsin B.
The optical properties of TPE-COOH and prodrug were
apoptotic probe (TPE-DEVD-RGD) (Scheme S2). GEM-induced
1
cell apoptosis will be initiated by the released GEM, and then then studied. It is well known that AIE fluorogen shows weak
DEVD peptide in TPE-DEVD-RGD will be cleaved by the fluorescence in good solvents but strong fluorescence in the
apoptosis activated caspase-3.5,9,17 Therefore, strong blue aggregated state. As is expected, TPE-COOH emitted strong
fluorescence will be observed because of the hydrophobic TPE blue fluorescence in DMSO/PBS mixture (1/199, v/v), while
residues with AIE characteristics. As a proof-of-concept, such prodrug
1 was almost non-fluorescent in the same medium on
cascade enzymatic reaction activatable GEM prodrug system account of its excellent solubility (Fig. 1b). The aggregation of
can be used as theranostic platform for killing pancreatic hydrophobic TPE-COOH in DMSO/PBS mixture (1/199, v/v) was
cancer cells and simultaneously for real-time and noninvasive confirmed by dynamic light scattering (DLS) with an intensity-
imaging of the therapeutic responses.
average hydrodynamic diameter of 825 nm (Fig. S9). This
cellular aggregation of TPE might be beneficial for enhanced
intracellular retention of the probe and thus for improving
tumor imaging in vivo.20 In view of these interesting optical
properties, prodrug
1 can be a potential specific light-up
apoptotic probe for therapeutic monitoring with minimal
background signals.
Scheme 1 Schematic illustration of cascade enzymatic reaction activatable
gemcitabine prodrug 1 with AIE-based intracellular light-up apoptotic probe for
in situ self-therapeutic monitoring of pancreatic cancer cells.
Carboxylated TPE (TPE-COOH, Scheme S1) was first
synthesized and confirmed by 1H NMR and electrospray
ionization-mass spectrum (ESI-MS) (Fig. S1, S2). Prodrug
1
(Scheme 1) was then custom-synthesized by ChinaPeptides Co.,
Ltd with more than 95% purity and confirmed by High
Performance Liquid Chromatography (HPLC) and ESI-MS
spectrum (Fig. S3, S4). Meanwhile, caspase-3-responsive GEM
prodrug
2 (Scheme S3, Fig. S5, S6) but without cathepsin B-
responsiveness, and cathepsin B-responsive GEM prodrug
3
(Scheme S4, Fig. S7, S8) but without caspase-3-responsiveness
were also custom-synthesized as controls.
Fig.1 (a) In vitro GEM release from prodrug
concentration of cathepsin B (0, 0.1, 0.3, 0.5 UN mL-1) and prodrug
with cathepsin B (0.5 UN mL-1). (b) Fluorescent emission spectra of prodrug
TPE-COOH in DMSO/PBS mixture (1/199, v/v). Inset: Corresponding digital
photos taken under irradiation by a UV lamp at 365 nm. (c) Fluorescent emission
spectra of prodrug (10μM) or prodrug (10μM) upon treatment with caspase-
(200 pM) in the presence or absence of Z-DEVD-FMK (20μM, caspase-3
inhibitor) and cathepsin
(0.5 UN mL-1). (d) Time-dependent fluorescent
emission spectra of prodrug (10μM) in the presence of caspase-3 (200 pM) in
DMSO/PBS mixture (1/199, v/v). (e) PL intensity of prodrug at 480 nm upon
addition of caspase-3 (200 pM) from 0 to 180 min. (f) PL intensity of prodrug
(10 μM) treated with various concentration of caspase-3 (0, 10, 25, 50, 100, and
200 pM) in DMSO/PBS mixture (1/199, v/v) for 60 min.
1
incubated with various
inbucated
and
2
1
For the prodrug delivery system, it is crucially important to
release original active drug for restoring its therapeutic
ability.5a,18 Since cathepsin B is overexpressed in pancreatic
cancer cells,19 the release behaviour of pharmaceutically active
1
3
3
B
1
1
1
GEM from prodrug
1
incubated with 0.5 UN mL-1 cathepsin B
was investigated by HPLC12b (Fig. 1a). Approximately 60% of
GEM was released in 6 h and close to 80% of GEM was
The light-up capability of prodrug
1 triggered by caspase-3
released after 24 h since GEM was conjugated to prodrug
cathepsin B-cleavable GFLG peptide sequence. Meanwhile, we
found that the release behaviour of GEM from prodrug is
1 by
was subsequently investigated. As shown in Fig. 1c, prodrug
1
was also non-fluorescent in DMSO/PBS mixture (1/199, v/v),
but strong fluorescence signals were recorded upon incubation
with 200 pM caspase-3. However, when pretreated with
1
cathepsin B-concentration dependent. However, there is
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caspase-3 inhibitor Z-DEVD-FMK, almost no fluorescence could intracellular light-up apoptotic probe for therapeutic
be observed in the presence of caspase-3, indicating the monitoring of BxPC-3 cells.
specific cleavage of DEVD peptide sequence in prodrug
severely inhibited. It is worth noting that, upon incubation
with 0.5 UN mL-1 cathepsin B, the fluorescence of prodrug
1 was
1
increased little, indicating hardly no AIE effect happened since
hydrophilcity of the prodrug changes little with an almost
unchanged basic hydrophilic segment of the prodrug. In
addition, prodrug
3 was almost non-fluorescent in DMSO/PBS
mixture (1/199, v/v) and the fluorescence spectrum remained
no change in the presence of caspase-3 since the DAAD
peptide sequence was stable and TPE kept in the molecularly
dissolved state under this condition.
Fig. 2 (a) Cell viability of BxPC-3 cells incubated with various concentrations of
GEM, prodrug prodrug and prodrug for 72 h. *P<0.05, **P<0.01,
compared to prodrug . (b) Caspase-3 expression in BxPC-3 cells incubated with
prodrug , prodrug , and prodrug at the concentration of 20 μM for 72 h.
1,
2
,
3
1
1
2
3
The fluorescence change of prodrug
1 upon treatment with
Finally, taking advantage of the intracellular cascade
enzymatic reaction, fluorescence microscopy was carried out
to study the capability of GEM-induced apoptosis in BxPC-3
cells by recording the intracellular fluorescence upon
caspase-3 was also recorded over time in DMSO/PBS mixture
(1/199, v/v). As shown in Fig. 2d, after incubation with
caspase-3, the fluorescence of prodrug
1
increased
dramatically. The fluorescence reached a plateau in 60 min
which was 22-fold higher than the intrinsic emission of
incubation with prodrug
1
, prodrug 2, or prodrug
3. As shown
in Fig. 3, upon incubation with 10
µ
M prodrug 1, strong blue
prodrug
the fluorescent emission of prodrug
As shown in Fig. 2f, with increasing concentrations of caspase-
3 from 0 to 200 pM, the fluorescence of prodrug increased
1
(Fig. 2e). The effect of caspase-3 concentration on
fluorescence was observed in BxPC-3 cells because of the
intracellular cascade enzymatic reaction generated
hydrophobic TPE residues with AIE characteristics. However, if
1
was further investigated.
1
incubated with prodrug
2, only weak fluorescence was
gradually, which might be ascribed to the increased amount of
hydrophobic TPE aggregates. These results confirmed that the
caspase-3 triggered release of TPE might be used as a specific
indicator of caspase-3 activity in the cells.
observed in BxPC-3 cells, since GEM could be hardly released
from the stable GAAG peptide sequence, leading to few cell
apoptosis, which was in accordance with the results in Fig. 2b.
Meanwhile, almost no fluorescence of prodrug
3 could be
In order to evaluate the therapeutic effect of prodrug
in vitro cell proliferation inhibition against human pancreatic
cancer cells BxPC-3 cells compared to free GEM, prodrug
and prodrug was studied using MTT (3-(4,5-dimethylthiazol-
1, the
monitored in cells due to the molecularly dissolved state of
TPE in TPE-DAAD-RGD probe that could not be specifically
cleaved by capase-3, which was in good agreement with FL
2,
3
results in Fig. 1c. These results strongly proved that prodrug
1
2-yl)-2,5-diphenyltetrazolium bromide) assay (Fig. 2a). After
incubation for 72h, the cell viabilities upon treatment with
was not only promising in targeted GEM delivery but also
potential as intracellular light-up apoptotic probe for in situ
self-therapeutic monitoring of pancreatic cancer cells.
prodrug
1
and prodrug
3
were almost the same but much
. This is because GFLG peptide
lower than that with prodrug
2
sequence of prodrug could be specifically cleaved by
cathepsin-B and active GEM would be thus released in BxPC-3
cells, which kills the cells, while GAAG could not. Interestingly,
high dose of prodrug
1 showed superior cell proliferation
inhibition compared to the same concentration of GEM,
probably owing to efficient cellular internalization of prodrug
and successful release of GEM from the prodrug.
1
GEM kills BxPC-3 cells by inducing cell apoptosis with
activated caspase-3,20 and caspase-3 plays an important role in
signal transforming of cell apoptosis.10 In order to verify
whether the caspase-3 could be activated by prodrug
1 in
BxPC-3 cells, the expression of caspase-3 was investigated
using Ac-DEVD-pNA, a fluorogenic caspase-3 substrate (Fig. 2b).
Compared to the control, BxPC-3 cells incubated with prodrug
2
only had a slight increase of the expression of caspase-3
because GEM cannot be effectively released. However, when Fig. 3 Fluorescence microscopy images of the prodrug
1 (A), prodrug 2 (B), and
3 (C) for 3 h. A1, B1, C1: blue fluorescence images of TPE; A2,B2,C2:
prodrug
bright field images; A3,B3,C3: the merge images.
incubated with prodrug
1 or prodrug 3, the expression of
caspase-3 increased dramatically owing to the effective
cathepsin B triggered GEM release, which was consistent with
the previous MTT results. These results suggested that BxPC-3
In summary, a theranostic GEM prodrug for targeted drug
delivery and evaluation of in situ therapeutic effect was
developed in this work. After cellular internalization of prodrug
cells could generate caspase-3 stimulated by prodrug
1 and
1, active GEM and the cell apoptotic probe (TPE-DEVD-RGD)
the generated caspase-3 could be used as specific initiator for
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a
,
,
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time and noninvasive imaging of the therapeutic responses.
On the other hand, the fluorescence of TPE is in the blue
spectrum, which strongly restricts the in vivo applications. In
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Financial supports from the National Natural Science
Foundation of China (51573160, 21574114), and the Scientific
Research Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry ([2015]311) are gratefully
acknowledged. There are no conflicts of interest to declare.
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