A unimolecular theranostic system with H2O2-specific response and AIE-activity for doxorubicin releasing and real-time tracking in living cells

Article abstract of DOI:10.1039/c8ra01185k

A theranostic drug delivery system composed of tetraphenyl-ethene (AIEgen), benzyl boronic ester (trigger), and doxorubicin (drug) was designed and synthesized; its utilities for cell imaging, drug delivery tracking, and cancer cell cytociding were evalua

Full text of DOI:10.1039/c8ra01185k

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A unimolecular theranostic system with H O -
2
2
specific response and AIE-activity for doxorubicin
releasing and real-time tracking in living cells†
Cite this: RSC Adv., 2018, 8, 10975
a
a
a
a
a
a
Xiaoying Gao, Jie Cao, Yinuo Song, Xiao Shu, Jianzhao Liu,* Jing Zhi Sun,
b
acd
Bin Liu * and Ben Zhong Tang*
Received 7th February 2018
Accepted 14th March 2018
A theranostic drug delivery system composed of tetraphenyl-ethene (AIEgen), benzyl boronic ester (trigger),
and doxorubicin (drug) was designed and synthesized; its utilities for cell imaging, drug delivery tracking,
and cancer cell cytociding were evaluated.
DOI: 10.1039/c8ra01185k
rsc.li/rsc-advances
Cancer is a disease that heavily threatens human lives and AIEgens to emit efficiently at high concentrations and in solid
chemotherapy is known to be an effective route for cancer state. Compared with conventional dyes with aggregation
1,2
treatment. To achieve good therapeutic efficiency, controlled caused quenching (ACQ) property, AIEgens have shown higher
drug delivery systems are usually used in chemotherapy. photo-bleaching resistance and emission stability. In addition,
Traditional drug delivery systems are nanocarriers that could they have shown lower cytotoxicity than inorganic quantum
specically respond to a certain physiological environment (e.g., dots. Thus they are ideal candidates for cell imaging and drug
1
3–16
redox, oxidation, pH, etc.), and release the drug at the same delivery tracking.
3–5
time. Recently, some theranostic drug delivery systems have
The application of AIEgens to real-time imaging and tracking
6–9
been developed and attracted broad attention. Theranostic drug release process emerged as a novel strategy. For example,
drug delivery systems that integrate diagnosis and therapy oen Liu et al. developed a light-up theranostic agent by tactfully
respond to cancer-associated stimuli, release the drug, and linking a silole-based AIEgen, a cyclic RGD cancer cell-targeting
simultaneously reveal an output signal change which can be peptide, a platinum Pt(IV) prodrug, and a stimulus responsive
6–9
17
tracked in a real time manner.
peptide together. The reductant of ascorbic acid in U87-MG
Fluorescent probing is one of the mostly used signaling cancer cells reduced the prodrug Pt(IV) to the active Pt(II) drug,
techniques to display signal change upon drug release. which triggered apoptosis by activating the pro-apoptotic
Recently, uorescent probes with AIE (aggregation-induced enzyme caspase-3. Subsequently, the enzyme cleaved the
emission) property, namely AIEgens, have attracted great peptide to release the AIEgens, which aggregated in the cyto-
attention in both fundamental research and industrial appli- plasm and emitted strongly to report the drug-induced
10–16
cations.
AIEgens are non-emissive when molecularly dis- apoptosis. Using this strategy, another light-up theranostic
solved in solution but can be induced to emit strongly in agent containing a tetraphenylethene (TPE) AIEgen, a RGD
aggregate state because of the restricted intramolecular peptide, a Pt(IV) prodrug and a stimulus-responsive peptide was
10–16
motions (RIM).
This unique RIM mechanism allows designed, and it showed evident AIE-characteristics and ex-
pected cytotoxicity for breast cancer cells over normal cells. In
a later design, both prodrugs of Pt(IV) and doxorubicin (DOX)
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, were introduced into the light-up theranostic agent, which
a
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou
enhanced the apoptosis of cancer cells by the synergistic effect
3
10027, China. E-mail: liujz@zju.edu.cn
18
of the two drugs. In 2014, Zou, Liang and colleagues reported
a uorescence based self-indicating drug delivery system, which
is capable of signaling spatiotemporal drug release with TPE
b
Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. E-mail: cheliub@
nus.edu.sg
5
c
and DOX composite nanoparticles (NPs). The emission of TPE
Department of Chemistry, Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, molecules in the composite NPs was partially quenched by the
China. E-mail: tangbenz@ust.hk
DOX aggregates via a uorescence resonance energy transfer
d
SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team,
(FRET) mechanism. Aer being taken up into lysosomes, the
State Key Laboratory of Luminescent Materials and Devices, South China University
of Technology, Guangzhou, 510640, China
low internal pH triggered the detachment of DOX from the
composite NPs and simultaneously enhanced the emission
†
Electronic supplementary information (ESI) available: Experimental procedures,
structure characterization data for the ABD system and particle size distribution of from AIEgens due to the absence of FRET process. This drug
the ABD system in buffer solution. See DOI: 10.1039/c8ra01185k
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delivery mode can track the sub-cellular location of the delivery ABD-system in hand, we rst investigated its uorescence
system and the drug releasing site. responses towards hydrogen peroxide H O , in order to examine
2
2
In this work, we developed a theranostic drug delivery system the proposed working mechanism shown in Scheme 1. The
different from those reported in the literature. The system is ABD-system itself in the solid state was weakly red emissive
constructed from an AIEgen (carboxylated TPE), a benzyl- under the excitation of UV light due to efficient FRET, in spite of
boronic ester (BBE), and a DOX prodrug. It is abbreviated as the strong emission of the TPE unit in solid state. In PBS buffer,
ABD-system, and its working mechanism is illustrated in almost no emission was recorded for the ABD-system upon
Scheme 1. In comparison with the previous work, a prime excitation at 330 nm (Fig. 1), because the uorescence of TPE is
difference in our design lies on the BBE trigger, which is absorbed by DOX, while DOX is weakly uorescent in aggregate
sensitive to hydrogen peroxide that is one of the cellular reactive state (the size distribution of the aggregates is shown in
oxygen species (ROS). ROS play key roles in controlling the Fig. S13†). Aer the addition of H O (100 mM) into buffer
2
2
function and health of cells, and alleviated ROS level has been solution, blue uorescence was observed and the brightness
19–24
correlated to cancerous cells.
With their high reactivity, enhanced gradually with time. As shown in Fig. 1A and B, the
several drug delivery systems utilized ROS-sensitive functional emission peak appeared at around 480 nm, which was assigned
groups, such as BBE and 2,4-dinitro-benzene sulfonyl, as trig- to the emission from the aggregates of hydrophobic carboxyl-
25–28
gers to activate prodrugs.
So far, cellular ROS has never been ated TPE molecules, as indicated by its AIE property (Fig. S15
used as the internal agent to trigger the drug release during and S16†). This is because, when the BBE unit is oxidized by
which a uorescence turn-on mechanism is utilized to monitor H O , the linkage between TPE and DOX is broken, and DOX
2
2
the progress in a real-time mode. When chemically bonded and carboxylated TPE are released (cf. Fig. S17†). The hydro-
29
together via a self-immolative BBE linker, the TPE and DOX phobic carboxylated TPE molecules form aggregates which emit
units are close to each other. Moreover, the emission maximum blue uorescence, while the DOX molecules are soluble in the
(480 nm) of TPE is largely over-lapped with the absorption buffer solution and emit weak red uorescence. Since the
maximum (480 nm) of DOX. As DOX is weakly emissive due to distance between carboxylated TPE and DOX is far enough,
its ACQ property, the ABD system is faintly emissive in solution FRET process is prohibited and hence the blue emission has
or in aggregate state because of the unique emission mecha- been observed.
nism of the AIEgen and the efficient FRET from TPE to DOX. If
the ABD systems are internalized into a living cell with elevated
cellular ROS level, the BBE linker would be cleaved, thereby the
DOX and TPE units would be disassociated with each other and
the FRET process is disrupted. As a result, DOX would be
released and taken up into cell nucleus with red uorescence.
Meanwhile, the TPE molecules would form aggregate in the
cellular plasma for their hydrophobic nature thereby the blue

uorescence could be detected. By monitoring the uorescence
changes in blue and red channels, the spatial and temporal
information of the DOX release could be extracted.
The chemical structure of the theranostic ABD-system is
shown in Scheme 1, and its synthetic route, preparation
procedure, and characterization data of related intermediates
are described in the Experiment section and ESI† (Fig. S1 to
S12†). The spectral characterization results indicated that the
expected ABD-system has been successfully obtained. With the
Fig. 1 (A) Changes in fluorescence (FL) spectra of the ABD-system (10
mM) in PBS buffer solution in the presence of H (100 mM) for
2 2
O
different times (0 to 90 min). (B) Plot of FL intensity versus incubation
time. The data are extracted from the spectra in (A). (C) FL spectra of
the ABD-system in PBS buffer solution incubated with different
concentrations of H
versus H concentration. The data are extracted from (C), and the
inset photographs show the FL images of the stock solutions with and
2 2
O for 90 min; (D) plot of the relative FL intensity
2 2
O
without H
2
O
2
. Concentration of the ABD-system (10 mM); PBS buffer
ꢀ
Scheme 1 Chemical structure of the theranostic drug delivery ABD- solution (pH ¼ 7.8, 10 mM, containing 1% DMSO); temperature: 37 C;
system and a schematic illustration of its working mechanism in a cell excitation wavelength: 330 nm for FL measurement and 365 nm for
with elevated hydrogen peroxide level.
photograph.
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As indicated by the experimental data shown in Fig. 1A and intensity was recorded in the spectral window of 550–650 nm.
B, the emission intensity increased gradually and the spectral The overlay (Fig. 3D) of the bright eld (Fig. 3C) with the uo-
features reached a steady state (unchanged) aer 80 min. The rescent images suggest that the ABD-system is uniformly
experimental results shown in Fig. 1C and D were obtained by distributed in the cytoplasm.
reacting with H
behavior is observed. The emission intensity continuously 5 mg mL phorbol-12-myristate-13-acetate (PMA) for 30 min
increases with the increasing H concentration from 10 to 90 and then incubated for another 2.5 hours, evident blue
2
O
2
for 90 min. A prominent dose-dependent
When ABD-system loaded HeLa cells were treated with
ꢁ
1
2 2
O
mM, and the photograph clearly displays the blue emission from emission was detected in spectral window of 420–540 nm
the AIEgen (inset of Fig. 1D). A uorescence intensity (Fig. 3E). Meanwhile, red emission was also collected in the
enhancement of 142-fold was observed when the H O
concentration was increased to 90 mM.
wavelength range of 550–650 nm (Fig. 3F). Since PMA is an
agent widely used to in situ induce the generation of H O in
2
2
2
2
Considering that other ROS, RNS (reactive nitrogen living cells, the introduction of PMA can effectively elevate
ꢁ
species) and RSS (reactive sulphur species), such as O
2
,
2 2
the H O level and thereby trigger the release of both
ꢁ
1
GSH, ClO , $OH,
alkylperoxyl radical (ROO$), NO
coexist with H in physiological environment, we also medium (e.g., cytoplasm), and hence formed aggregates. Due
2
O , tert-butyl hydroperoxide (TBHP), carboxylated TPE and DOX in the cytoplasm. The generated
ꢁ
ꢁ
ꢁ
2
, NO
3
, and S
2
, may carboxylated TPE molecules were insoluble in aqueous
2
O
2
examined the uorescent response of the ABD-system to the to the inhibited FRET process induced by DOX leaving and
interfering species. As shown in Fig. 2, except for H O , all of the TPE aggregate formation, strong blue uorescence from
2
2
the other reactive species can only lead to very small changes TPE donor was recorded according to the RIM mechanism. At
in uorescence intensity under the identical experimental the same time, DOX molecules were also released into cyto-
condition. Such an excellent selectivity is ascribed to the plasm. The merged image indicates that the blue and red
rational molecular design, since among the ROS we tested, emissions from the aggregates of the carboxylated TPEs and
only H
2 2
O can react with the BBE unit to induce the separa- DOXs have good overlap and are distributed all over the
tion between TPE and DOX units and then turn on uores- cytoplasm.
cence emission.
Aer further incubation for additional 3 h, the blue emission
Based on the above results, the ABD system works well in still existed in the cytoplasm (Fig. 3I), while the red emission
buffer solutions. The blue emission from the aggregate of could be observed in the cell nucleus (Fig. 3J). This indicates
carboxylated TPE can be specically triggered by H O , indi- that some DOX molecules are translocated into cell nucleus.
2
2
cating the releasing of DOX. To check whether this system can This observation is reasonable because DOX is a drug that
work well in living cells, we incubated HeLa cells in different works on DNA. In the overlay image (Fig. 3L), the entities with
conditions and monitored the uorescence from the cells using purple uorescence are assigned to cell nucleus, indicative of
confocal laser scanning microscopy (CLSM) technique. When the colour mixing of blue and red emissions. The surrounding
HeLa cells were loaded with 10 mM ABD-system and incubated
ꢀ
at 37 C for 2 h, no uorescence emission could be recorded in
blue channel (420–540 nm), but red emission with moderate
Fig. 2 FL response of the ABD system (10 mM) to a series of ROS (100 mM) Fig. 3 Confocal images of HeLa cells after incubation with TPE–DOX
in PBS buffer solution. I
0
and I are the FL intensity of the ABD-system in with different treatment; (A–D) no more treatment; (E–H) cells treated
with PMA for 30 min and incubated for another 2.5 hour; (I–L) cell
PBS buffer solution in the presence of ROS, RNS, and RSS including H
O ,
2 2
ꢁ
ꢁ
1
ꢁ
ꢁ
ꢁ
O
2
, GSH, ClO , $OH, O
2
, TBHP, ROO$, S
2
, NO
2
and NO
3
. PBS buffer treated with PMA for 30 min and incubated for another 5.5 hour. Blue
ꢀ
solution (pH ¼ 7.8, 10 mM, containing 1% DMSO); temperature: 37 C; channel: excitation wavelength: 405 nm; collection wavelength: 420–
excitation wavelength: 330 nm; reaction time: 100 min.
540; red channel: excitation wavelength: 488 nm; collection wave-
length: 550–650 nm. All images share the same scale bar (20 mm).
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blue uorescence comes from the aggregated carboxylated In addition, the ABD system shows high stability in the pH
TPEs. range from 4 to 9 and good selectivity over other interfering
In order to evaluate therapeutic performance of the ABD reactive species including OCl , O , $OH, GSH, O , TBHP,
ꢁ
ꢁ
1
2
2
ꢁ
ꢁ
ꢁ
system, cell viability was studied using MTT (3-(4,5-dimethyl- ROO$, NO2 , NO3 and S2 , which are also involved in biolog-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown ical systems.
in Fig. 4, without the ABD system, the cell viability is as high as
9
8% in the presence of PMA under standard incubation
Conclusions
condition. PMA itself has no therapeutic effect on HeLa cells.
Without the treatment of PMA, the presence of ABD system
could not induce the therapeutic effect, and the cell viabilities
in different concentrations and incubation time were almost
the same, as suggested by the data in Fig. 4. In sharp contrast,
when activated with PMA, cell viabilities decreased dramatically
and synergistically with the increment of prodrug concentration
and incubation time. As a direct evidence, the bright eld
images (Fig. 3C, G and K) and the confocal uorescent images
demonstrate that the morphologies of HeLa cells changed
signicantly upon treated with ABD and PMA for hours. These
In short, the ABD system demonstrates multiple functions of

uorescent cell imaging, drug releasing and drug delivery
process tracking, and thus it is a novel and promising candidate
for theranostic drug delivery system.
Conflicts of interest
There are no conicts to declare.
results strongly prove that ABD system has good therapeutic Acknowledgements
effects on HeLa cells.
This work was nancially supported by the National Science
In summary, an ABD-system consisting of carboxylated TPE
AIEgen), active linker (benzyl-boronic ester) and DOX (drug)
Foundation of China (51573158, 21788102), the key project of
Ministry of Science and Technology of China (2013CB834704),
the Innovation and Technology Commission (ITC-CNERC-
(
moieties has been designed, synthesized and characterized. Its
drug releasing and uorescent tracking processes and its ther-
apeutic effect have been studied. In living HeLa cells incubated
with the ABD-system, sole red emission can be observed. Aer
treatment with PMA, H O is generated in situ and it reacts with
the benzyl-boronic ester moiety, leading to the decomposition
of the ABD system and the spatial separation of carboxylated
TPE and DOX moieties. As a result, both of the blue emission
from the AIEgen and red emission from DOX molecules have
been monitored in the respective spectral channels, due to
restricted FRET between AIEgens and DOX. Based on the dual
emission colors, the DOX releasing can be monitored. Aer
a short period of time, the released DOX molecules enter into
nucleus to realize its therapeutic function, which is revealed by
the observation of the red emission in the nucleus region. The
therapeutic effect has been estimated by the MTT experiments.
14SC01), and the Research Grants Council of Hong Kong
(16301614, 16305015, 16308016, N_HKUST604/14 and A-
HKUST605/16). J. L. thanks the Thousand Young Talents Plan
of China and Hundred Talents Program of Zhejiang University.
B. Z. T. is also grateful for the support from the Science and
Technology Plan of Shenzhen (JCYJ201602229205601482), B. L.
thanks the National University of Singapore (R279-000-482-133)
for nancial support.
2
2
Notes and references
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