A targeted theranostic platinum(iv) prodrug containing a luminogen with aggregation-induced emission (AIE) characteristics for in situ monitoring of drug activation

Article abstract of DOI:10.1039/c3cc49516g

A targeted theranostic platinum(iv) prodrug based on a luminogen with aggregation-induced emission (AIE) characteristics was developed for selective and real-time monitoring of drug activation in situ. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc49516g

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A targeted theranostic platinum(IV) prodrug
containing a luminogen with aggregation-induced
emission (AIE) characteristics for in situ
monitoring of drug activation†
Cite this: Chem. Commun., 2014,
50, 3868
Received 16th December 2013,
Accepted 19th February 2014
Youyong Yuan,‡a Yilong Chen,‡b Ben Zhong Tang*bc and Bin Liu*ad
DOI: 10.1039/c3cc49516g
www.rsc.org/chemcomm
A targeted theranostic platinum(IV) prodrug based on a luminogen with and bioimaging.4 They are non-emissive in the molecularly dissolved
aggregation-induced emission (AIE) characteristics was developed for state, but induced to emit bright fluorescence by aggregation due to
selective and real-time monitoring of drug activation in situ.
the restriction of intramolecular rotations (RIR) and prohibition of
energy dissipation via non-radiative channels.5 Recently, we have
developed AIE luminogen based light-up probes for monitoring of
cellular molecules.6 However, reports describing theranostic drug
conjugate based on these molecules have not yet been published.
In this communication, we report the synthesis and biological
evaluation of a targeted theranostic platinum(IV) prodrug delivery
system based on an AIE luminogen for in situ monitoring of the
platinum(IV) prodrug activation. The theranostic system is composed
of a chemotherapeutic prodrug Pt(IV), which can be reduced to active
Pt(II) inside the cells, a tetraphenylethene pyridinium (PyTPE) unit
with AIE characteristics, a short hydrophilic peptide with five
aspartic acid (D5) units to ensure its water solubility6b and a cyclic
arginine–glycine–aspartic acid (cRGD) tripeptide as a targeting ligand
(Scheme 1). The prodrug is highly water-soluble and can accumulate
preferentially in cancer cells that overexpress avb3 integrin. In
aqueous media, the AIE moiety is non-fluorescent due to the high
hydrophilicity of the D5-cRGD, but its emission is enhanced signifi-
cantly after the reduction of the Pt(IV) complex to active Pt(II) drug.
The fluorescence enhancement (‘‘turn-on’’) is attributed to the
restriction of intramolecular rotations of the PyTPE phenyl rings in
the cleaved residues, which populates the radiative decay channels.
Our prodrug design offers good opportunity for efficient platinum
Cisplatin has been widely used in clinics to treat a broad range of
human malignancies. However, like most other cytotoxic anti-
cancer drugs, its clinical use is often limited due to severe side
effects.1 To address these limitations, researchers have developed a
promising alternative strategy by using non-toxic Pt(IV) prodrugs,
which can be intracellularly activated by reduction to Pt(II) complex
and restore their latent cytotoxic activity.2 Although the use of Pt(IV)
prodrug has been proved to be successful in improving the efficacy
of platinum-based drugs, it remains a challenge to elucidate how
and when Pt(IV) prodrugs are reduced after cell internalization.
To date, fluorescent techniques remain the most effective strategy
for monitoring the intracellular reduction process of platinum(IV) com-
plex.3 The most direct method in studying platinum reduction is by
attaching fluorophore–quencher pairs to the two ligands of Pt(IV) com-
plexes, which can restore the fluorescence due to prodrug reduction
induced donor–acceptor separation.3a–c However, very few systems
have been successfully utilized in living cell imaging.3d,e It is highly
desirable that one be able to design and develop a theranostic system
which can specifically deliver the Pt(IV) prodrugs and simultaneously
perform real-time monitoring of the drug activation in situ, which can
provide a quantitative readout of dynamic processes in living cells.
Luminogens with aggregation-induced emission (AIE) charac-
teristics have offered a powerful and versatile tool for biosensing
a Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore 117576. E-mail: cheliub@nus.edu.sg
b Department of Chemistry, Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
E-mail: tangbenz@ust.hk
c SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team,
State Key Laboratory of Luminescent Materials and Devices,
South China University of Technology, Guangzhou, 510640, China
d Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602
† Electronic supplementary information (ESI) available: Detailed experimental
methods and additional data. See DOI: 10.1039/c3cc49516g
‡ These authors contribute equally.
Scheme 1 Schematic illustration of prodrug PyTPE-Pt-D5-cRGD design
strategy and the fluorescence turn-on monitoring of drug activation.
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drug delivery and real-time monitoring of the drug activation with a
high signal-to-noise ratio.
Amine-functionalized PyTPE (PyTPE-NH2) was synthesized by redu-
cing azide-PyTPE (PyTPE-N3) in methanol (Scheme S1 and Fig. S1
and S2, ESI†). Pentafluorophenol-activated Pt(IV) complex was prepared
from commercially available anticancer drug cisplatin and was used as
the linker (Scheme S2 and Fig. S3–S5, ESI†). The synthetic route for the
prodrug PyTPE-Pt-D5-cRGD is shown in Scheme S3 (ESI†). Asymmetric
functionalization of activated Pt(IV) complex with PyTPE-NH2
and amine-functionalized peptide D5-cRGD in the presence of
N,N-diisopropylethylamine afforded the prodrug in 42% yield.
A control prodrug PyTPE-Pt-D5 with a similar structure but without
cRGD moiety was also synthesized in 44% yield (Scheme S4, ESI†). In
addition, a non-activatable control PyTPE-C6-D5-cRGD was prepared
in 46% yield by using disuccinimidyl suberate to replace activated
Pt(IV) complex in the coupling reaction (Scheme S5, ESI†). The NMR
and MS characterization confirmed the right structures of the
compounds with high purity (Fig. S6–S14, ESI†).
Fig. 1 (A) HPLC chromatograms showing the reaction of DDTC with cisplatin
and reduced Pt(IV) prodrug: (1) DDTC alone; (2) the reaction between DDTC
and cisplatin; (3) the reaction between DDTC and PyTPE-Pt-D5-cRGD; (4) the
reaction between DDTC and PyTPE-Pt-D5-cRGD (10 mM) in the presence of
ascorbic acid (1 mM) for 12 h. (B) PL spectra of PyTPE-NH2 and PyTPE-Pt-D5-
To evaluate PyTPE-Pt-D5-cRGD as a potential anticancer drug, we
first studied the nature of the formed Pt(II) species upon reduction. cRGD in DMSO/PBS mixtures (v/v = 1/199). Inset: the corresponding photo-
graphs taken under illumination of a UV lamp at 365 nm. (C) Time-dependent
Previous studies have shown that diethyldithiocarbamate (DDTC)
PL spectra of PyTPE-Pt-D5-cRGD (10 mM) treated with ascorbic acid (1 mM).
(D) Plot of PL intensity at 605 nm versus concentrations of PyTPE-Pt-D5-
cRGD with the incubation of ascorbic acid (1 mM) for 75 min in DMSO/PBS
can react with Pt(II) complexes to yield the adduct of Pt(DDTC)2 but
not with the stable Pt(IV) complexes.7 LC-mass was subsequently
used to monitor the adduct formation between Pt(II) and DDTC.
Ascorbic acid was selected because it has been reported to be one of
(v/v = 1/199). Data represent mean values Æ standard deviation, n = 3.
the major reducing agents for the reduction of Pt(IV) in cells.2b As the prodrug itself. The non-targetable prodrug PyTPE-Pt-D5 shows a
shown in Fig. 1A, free DDTC has a retention time of 13.3 min, which similar fluorescence intensity increase after incubation under the
is slightly shorter than that of Pt(DDTC)2 (14.4 min). In the absence same conditions (Fig. S18A, ESI†). In contrast, negligible fluorescence
of ascorbic acid, there is no peak formed for Pt(DDTC)2 when intensity increase is observed for PyTPE-C6-D5-cRGD (Fig. S18B, ESI†).
PyTPE-Pt-D5-cRGD is incubated with DDTC. Only in the presence of Further titration of PyTPE-Pt-D5-cRGD with other bio-acids and
ascorbic acid, the prodrug can react with DDTC to form Pt(DDTC)2 proteins showed negligible fluorescence intensity change, indicating
with a mass-to-charge ratio (m/z) of 492.104 (Fig. S15, ESI†), high stability of the prodrug (Fig. S19, ESI†). Only in the presence of
confirming that the released Pt entities are indeed Pt(II) species. the reducing agent (glutathione or ascorbic acid), the prodrug showed
Another piece of evidence is that a peak at 14.1 min with a m/z of an intense fluorescence change. These results indicate that the
1140.344 (Fig. S16, ESI†) is found for PyTPE-COOH (Scheme S6, fluorescence enhancement is attributed to the reduction of the
ESI†) after reduction. These results indicate that the prodrug can be prodrug with the release of AIE residue (PyTPE-COOH).
reduced by ascorbic acid to generate the active Pt(II) drug and
release the axial moieties simultaneously.
Next, we incubated different concentrations of PyTPE-Pt-D5-
cRGD with ascorbic acid (1 mM) and the fluorescence intensities
The UV-vis absorption spectra of PyTPE-NH2 in THF and PyTPE- of the prodrug were monitored. The plot of the PL intensities at
Pt-D5-cRGD in DMSO/PBS (phosphate buffered saline) mixtures 605 nm against the prodrug concentration is perfectly linear (Fig. 1D
(v/v = 1/199) are shown in Fig. S17 (ESI†). Both have a similar and Fig. S20, ESI† for PyTPE-Pt-D5), suggesting the possibility of
absorption profile in the range of 348–500 nm with a maximum at quantification of the activated Pt(II) drugs. The gradually enhanced
405 nm. The photoluminescence (PL) spectra of PyTPE-NH2 and fluorescence intensities are due to the increased amounts of PyTPE
PyTPE-Pt-D5-cRGD in DMSO/PBS (v/v = 1/199) are shown in Fig. 1B. aggregates formed in aqueous media. The molecular dissolution of
The hydrophobic PyTPE-NH2 shows intense fluorescence while the probe and the aggregate formation of the cleaved products were
PyTPE-Pt-D5-cRGD is almost non-fluorescent in the same mixture, confirmed by laser light scattering (LLS) measurements. In the
due to easy intramolecular rotations of the TPE phenyl rings in aqueous mixture, no LLS signals could be detected from the
aqueous media. The significant difference in the PL intensities of solution of PyTPE-Pt-D5-cRGD. However, after the reduction,
PyTPE-NH2 and PyTPE-Pt-D5-cRGD offers opportunity for the pro- the residual hydrophobic AIE luminogen clusters into aggregates
drug to be used for real-time monitoring of the drug activation.
with an average diameter of 145 nm (Fig. S21, ESI†). Therefore, the
To study the response of the prodrug upon reduction, we incu- drug activation process can be easily monitored on the basis of the
bated PyTPE-Pt-D5-cRGD (10 mM) with ascorbic acid (1 mM) in PL intensity changes.
DMSO/PBS (v/v = 1/199), and the fluorescence spectra were measured
The cell lysates of breast cancer cells MDA-MB-231 were directly
at different time points. As shown in Fig. 1C, the emission intensity of incubated with PyTPE-Pt-D5-cRGD (10 mM) and the fluorescence
PyTPE-Pt-D5-cRGD increases significantly with time, reaching the intensity at 605 nm was monitored over time. As shown in Fig. S22
plateau in 1.5 h, which is 18-fold higher than the emission from (ESI†), the fluorescence intensity increases quickly in a similar way to
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Fig. 3 Cell viability of MDA-MB-231 (A) and MCF-7 (B) cells upon treat-
ment with PyTPE-Pt-D5-cRGD, PyTPE-Pt-D5 and PyTPE-C6-D5-cRGD at
different concentrations for 72 h. Data represent mean values Æ standard
deviation, n = 3.
In conclusion, we report the synthesis and biological applications
of a fluorescent light-up prodrug based on an AIE luminogen for real-
time monitoring of drug activation inside the cells. Thanks to the
unique nature of the AIE luminogen, the prodrug is non-fluorescent
in aqueous media but becomes highly emissive when reduced inside
the cells. Similar to the cRGD functionalized prodrug which allows for
selective targeting of avb3 integrin on many angiogenic cancers,
replacing cRGD with other tumor receptor specific ligands will be
likely to yield more functionalized prodrugs for selective killing of
different cancer cells. The capability of monitoring prodrug activation
processes through fluorescence changes of AIE fluorogen offers
a new opportunity for the development of theranostic anti-
cancer therapeutics.
Fig. 2 Confocal images of MDA-MB-231 (A–F) and MCF-7 (G–I) cells after
incubation with PyTPE-Pt-D5-cRGD for 1 h (A), 2 h (B), 4 h (C), 6 h (D, G);
PyTPE-Pt-D5 for 6 h (E, H) and PyTPE-C6-D5-cRGD for 6 h (F, I). The nuclei
were stained with Hoechst 33342. All images share the same scale bar (20 mm).
that of the solution study with ascorbic acid in Fig. 2C. Meanwhile,
the fluorescence intensity shows a minimum change after incuba-
tion PyTPE-C6-D5-cRGD with the lysate, indicating that the prodrug
is highly stable in the environment containing cellular proteins.
To explore the capability of using PyTPE-Pt-D5-cRGD to monitor
intracellular drug reduction in cancer cells, the prodrug was incubated
with MDA-MB-231 and MCF-7 breast cancer cells. The confocal
imaging results are shown in Fig. 2. MDA-MB-231 cells with over-
expressed integrin avb3 on the cellular membrane were chosen as
integrin-positive cancer cells, while MCF-7 cells with a low level of
integrin avb3 expression were used as the negative control. As shown
in Fig. 2, after the incubation with PyTPE-Pt-D5-cRGD, the fluorescence
in MDA-MB-231 cells is increased gradually with time and reaches a
plateau in 6 h, whereas for MCF-7 cells only a weak fluorescent signal
could be detected even after 6 h incubation. In contrast, PyTPE-Pt-D5
displays weak fluorescence for both cell lines (Fig. 2E and H), which
might be due to the inefficient cellular uptake of both cells. These
results further strengthen the targeting ability of PyTPE-Pt-D5-cRGD.
When MDA-MB-231 cells were pre-treated with free cRGD prior to
We thank the Singapore National Research Foundation
(R-279-000-390-281), the Ministry of Defense (R279-000-340-232),
the Research Grants Council of Hong Kong (HKUST2/CRF/10 and
N_HKUST620/11), and the JCO (IMRE/12-8P1103).
Notes and references
1 V. Pinzani, F. Bressolle, I. J. Haug, M. Galtier, J. P. Blayac and P. Balmes,
Cancer Chemother. Pharmacol., 1994, 35, 1–9.
2 (a) X. Y. Wang and Z. J. Guo, Chem. Soc. Rev., 2013, 42, 202–224; (b) N. Graf
and S. J. Lippard, Adv. Drug Delivery Rev., 2012, 64, 993–1004.
3 (a) E. J. New, R. Duan, J. Z. Zhang and T. W. Hambley, Dalton Trans.,
2009, 3092–3101; (b) C. Molenaar, J. M. Teuben, R. J. Heetebrij,
H. J. Tanke and J. Reedijk, JBIC, J. Biol. Inorg. Chem., 2000, 5, 655–665;
(c) J. J. Wilson and S. J. Lippard, Inorg. Chim. Acta, 2012, 389, 77–84;
(d) Y. Song, K. Suntharalingam, J. S. Yeung, M. Royzen and S. J. Lippard,
Bioconjugate Chem., 2013, 24, 1733–1740; (e) D. Montagner, S. Q. Yap
and W. H. Ang, Angew. Chem., Int. Ed., 2013, 52, 11785–11789.
PyTPE-Pt-D5-cRGD incubation, the image shows weak fluorescence 4 (a) D. Ding, K. Li, B. Liu and B. Z. Tang, Acc. Chem. Res., 2013, 46,
2441–2453; (b) C. W. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Lam and
B. Z. Tang, J. Am. Chem. Soc., 2013, 135, 62–65; (c) J. Geng, K. Li, W. Qin,
L. Ma, G. G. Gurzadyan, B. Z. Tang and B. Liu, Small, 2013, 9, 2012–2019;
(Fig. S23, ESI†). The marked difference reveals that the selective uptake
of PyTPE-Pt-D5-cRGD by MDA-MB-231 cells is due to the integrin
receptor-mediated process. For PyTPE-C6-D5-cRGD, almost no detect-
able fluorescence was observed after 6 h incubation (Fig. 2F and I).
We next studied the cytotoxicity profile of the prodrug to MDA-
MB-231 and MCF-7 cells by a MTT assay. As shown in Fig. 3A,
PyTPE-Pt-D5-cRGD shows obvious cytotoxicity to MDA-MB-231 cells,
while in a parallel experiment for MCF-7 cells, it shows minimum
toxicity. The half-maximal inhibitory concentration (IC50) of PyTPE-
Pt-D5-cRGD towards MDA-MB-231 cells is 30.2 mM. In addition,
PyTPE-Pt-D5 and PyTPE-C6-D5-cRGD do not show obvious cytotoxi-
city to both cells. As MDA-MB-231 and MCF-7 cells were reported to
show a similar IC50 value toward cisplatin,8 these results indicate
that the cRGD moiety is able to guide the prodrug to avb3 integrin
overexpressed tumor cells, resulting in selective cell death.
(d) X. H. Huang, X. G. Gu, G. X. Zhang and D. Q. Zhang, Chem. Commun.,
2012, 48, 12195–12197; (e) Z. Wang, S. Chen, J. W. Y. Lam, W. Qin, R. T. K.
Kwok, N. Xie, Q. Hu and B. Z. Tang, J. Am. Chem. Soc., 2013, 135, 8238–8245;
( f) F. F. Wang, J. Y. Wen, L. Y. Huang, J. J. Huang and O. Y. Jin, Chem.
Commun., 2012, 48, 7395–7397; (g) T. Y. Han, X. Feng, B. Tong, J. B. Shi,
L. Chen, J. G. Zhi and Y. P. Dong, Chem. Commun., 2012, 48, 416–418.
5 (a) Y. N. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Commun., 2009,
4332–4353; (b) Y. N. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Soc.
Rev., 2011, 40, 5361–5388.
6 (a) H. B. Shi, R. T. K. Kwok, J. Z. Liu, B. G. Xing, B. Z. Tang and B. Liu,
J. Am. Chem. Soc., 2012, 134, 17972–17981; (b) Y. Y. Yuan, R. T. K.
Kwok, G. X. Feng, J. Liang, J. L. Geng, B. Z. Tang and B. Liu,
Chem. Commun., 2014, 50, 295–297.
7 J. Li, S. Q. Yap, C. F. Chin, Q. Tian, S. L. Yoong, G. Pastorin and
W. H. Ang, Chem. Sci., 2012, 3, 2083–2087.
8 S. M. Lee, T. V. O’Halloran and S. T. Nguyen, J. Am. Chem. Soc., 2010,
132, 17130–17138.
3870 | Chem. Commun., 2014, 50, 3868--3870
This journal is ©The Royal Society of Chemistry 2014