In Situ Monitoring Apoptosis Process by a Self-Reporting Photosensitizer

Article abstract of DOI:10.1021/jacs.9b00636

Although photodynamic therapy (PDT) has thrived as a promising treatment, highly active photosensitizers (PSs) and intense light power can cause treatment overdose. However, extra therapeutic response probes make the monitoring process complicated, ex situ and delayed. Now, this challenge is addressed by a self-reporting cationic PS, named TPE-4EP+, with aggregation-induced emission characteristic. The molecule undergoes mitochondria-to-nucleus translocation during apoptosis induced by PDT, thus enabling the in situ real-time monitoring via fluorescence migration. Moreover, by molecular charge engineering, we prove that the in situ translocation of TPE-4EP+ is mainly attributed to the enhanced interaction with DNA imposed by its multivalent positive charge. The ability of PS to provide PDT with real-time diagnosis help control the treatment dose that can avoid excessive phototoxicity and minimize potential side effect. Future development of new generation of PS is envisioned.

Full text of DOI:10.1021/jacs.9b00636

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Communication
In-Situ Monitoring Apoptosis Process by A Self-Reporting Photosensitizer
Tianfu Zhang, Yuanyuan Li, Zheng Zheng, Ruquan Ye, Yiru
Zhang, Ryan T. K. Kwok, Jacky W. Y. Lam, and Ben Zhong Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b00636 • Publication Date (Web): 28 Mar 2019
Downloaded from http://pubs.acs.org on March 28, 2019
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In-Situ Monitoring Apoptosis Process by A Self-Reporting
Photosensitizer
Tianfu Zhang,†,§ Yuanyuan Li,†,§ Zheng Zheng,† Ruquan Ye,∥ Yiru Zhang,‡ Ryan T. K. Kwok,† Jacky
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W. Y. Lam,† and Ben Zhong Tang,*,†,‡
† Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and
Reconstruction, Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Life Science and State
Key Laboratory of Molecular Neuroscience, Department of Chemical and Biological Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
‡ Center for Aggregation-Induced Emission, State Key Laboratory of Luminescent Materials and Devices, SCUT-HKUST
Joint Research Institute, South China University of Technology, Guangzhou 510640, China
∥ Department of Chemistry, City University of Hong Kong, Hong Kong, China
Supporting Information Placeholder
highly active PS and high power light can destroy normal cells
and compromise therapeutic efficacy.12,13 Hence, how to monitor
PS work in the body and how to evaluate the exact treatment end
point are important.
ABSTRACT: Although photodynamic therapy (PDT) thrived as
a promising treatment, highly active photosensitizers (PSs) and
intense light power can cause treatment overdose. However, extra
therapeutic response probes always make the monitoring process
complicated, ex-situ and delayed. Now this challenge is addressed
It is generally recognized that apoptosis can be induced by
PDT. Thus, apoptosis sensing can provide valuable diagnosis
information on the therapeutic efficacy.14,15 The most common
approach is monitoring the phosphatidylserine in early apoptotic
cells by fluorescent dye-labeled annexin V coupled with nuclear
stains, usually combining with flow cytometry.16,17 Although this
technique provides quantitative data, its temperature sensitivity,
extracellular Ca2+ concentration and operational demanding make
it impractical for in-situ and real-time monitoring of the apoptosis
process. To address this problem, fluorescent probes with
aggregation-induced emission (AIE) characteristics18 were first
developed by Tang19 for real-time drug evaluation and apoptosis
monitoring.20–22 On the other hand, Hong et. al reported a
positively charged AIE luminogen (AIEgen) for nucleus staining
in dead cell due to its entry to the fragmented nucleus and bound
to DNA electrostatically.23 Although nonemissive in solution
state, emission of AIEgen is activated by interacting with DNA
due to the restriction of intramolecular rotations (RIR) to reduce
the nonradiative decay.24–26 However, all the reported probes
serve only as the drug reporter and extra PSs are needed in the
therapeutic process. To the best of our knowledge, highly efficient
PSs enabling real-time and in situ evaluation of its therapeutic
effect remain challenging. In most biological systems, some
apoptosis inducing factors will undergo translocation from
mitochondria to the nucleus during apoptosis,27,28 accompanying
with mitochondrial depolarization,29 membrane permeabilization
and DNA fragementation.15 Imitating this intelligent biological
process, we worked on the design of new AIEgens for PDT
monitoring system. The new molecule called TPE-4EP+ exhibits
extremely high 1O2 generation efficiency. In addition, it undergoes
mitochondria-to-nucleus translocation during apoptosis induced
by PDT, enabling the construction of a real-time self-reporting
system to monitor the PDT process in situ. In comparison,
through adjusting the electrostatic interaction between molecules
and DNA, double and triple positively charged AIEgens possess
by
a self-reporting cationic PS, named TPE-4EP+, with
aggregation-induced emission characteristic. The molecule
undergoes mitochondria-to-nucleus translocation during apoptosis
induced by PDT, thus enabling the in-situ real-time monitoring
via fluorescence migration. Moreover, by molecular charge
engineering, we prove that the in-situ translocation of TPE-4EP+
is mainly attributed to the enhanced interaction with DNA
imposed by its multi-valent positive charge. The ability of PS to
provide PDT with real-time diagnosis help control the treatment
dose that can avoid excessive phototoxicity and minimize
potential side effect. Future development of new generation of PS
is envisioned.
Cancers, especially malignant and cancerous ones, have
become the leading causes of death worldwide.1 The most
common traditional cancer treatments, such as chemotherapy2 and
radiation therapy,3 are adopted with a “to be safe” overdose
strategy to ensure the complete removal of cancer cells and
better therapeutic effect. However, drug overdose and excessive
radiation always induce side effect and lesion of normal tissues.
Even worse, it can increase the risk of developing cancer
resistance and second cancers, and aggravate the situation.4,5
Therefore, it is highly desirable to timely monitor the therapeutic
responses in situ and to develop a more effective and noninvasive
treatment method to avoid excessive damage.
Photodynamic therapy (PDT) emerges as a promising treatment
modality thanks to its selective cytotoxicity towards cancer cells
with high spatiotemporal precision and noninvasive nature.6–8 The
principle of PDT is that a photosensitizer (PS) is activated under
light irradiation to generate toxic reactive oxygen species (ROS)
such as singlet oxygen (1O2) to kill cancer cells.9–11
Unfortunately, PDT shows also some drawbacks. For example,
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PDT effect, but no clear translocation signal and apoptosis
electrostatic interaction between negative transmembrane
potential of mitochondria within cancer cell and positively
charged pyridinium moiety, which restricts the intramolecular
rotation of molecules and boosts emission.33–35 In contrast, the
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monitoring ability were synthesized as control. With diagnostic
information provided from the self-reporting photodynamic
therapy system, fine control on the treatment time is possible to
avoid phototoxicity overdose and reduce potential side effect.
normal-cell-staining
experiments
exhibited
negligible
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fluorescence, which suggests the molecules have extraordinary
selectivity for cancer cells staining (Figure S8-S9). Correlate
experiments were also provided that selective accumulation of
TPE-4EP+ in cancer cells is due to the higher mitochondria
membrane potential of cancer cells over normal cells (Figure
S10–S11).36
Surprisingly, when we subsequently evaluated the
photostability of three molecules by continuous laser excitation,
only TPE-4EP+ was observed to redistribute from mitochondrial
to nucleus gradually (Figure S12–S16). We further investigated
the phenomenon by performing cell imaging of TPE-4EP+ with
white light exposure. Originally, TPE-4EP+ stained the
mitochondria exclusively (Figure 2). However, after 10 min of
white light illumination, TPE-4EP+ mainly redistributed in
nucleus while the area of mitochondrial seemed fragmented and
showed negligible fluorescent signal. Furthermore, cell shrinkage,
membrane blebbing and morphology change can be visualized in
the bright field images, which are the traits of cell apoptosis.
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Figure 1. (A) Molecular structures of TPE-2EP+, TPE-3EP+ and
TPE-4EP+. (B) Proposed Mechanism of mitochondria-to-nucleus
translocation of TPE-4EP+.
To investigate the charge effect, we synthesized a series of
fluorogens: TPE-2EP+, TPE-3EP+ and TPE-4EP+ by adjusting
the number of pyridinium moiety (Figure 1A).30 Their chemical
structures and optical properties were characterized and
summarized (Scheme S1–S2, Figure S1–S5, Table S1). Because
of their ionic character, all the molecules exhibited good water-
solubility and weak emission. After increasing the fraction of
EtOH as the poor solvent, the emission intensities gradually
increased (Φ = 15%, peak emission = 610 nm) due to RIR when
aggregate formation. Clearly, three molecules possess AIE
characteristics.
Figure 3. Real-time confocal imaging of HeLa Cells under
continuous 405 laser irradiation stained with (A−D) TPE-4EP+
(upper panel). Real-time confocal imaging of HeLa Cells under
the same condition stained with TPE-4EP+, followed by the
addition of (E−H) Annexin V-FITC and (I−L) PI.
Aiming to testify the mechanism of fluorescence translocation
phenomenon, FITC-annexin V and propidium iodide (PI) assay is
used to identify early and late stage apoptotic versus necrosis. The
real-time imaging of TPE-4EP+ was performed (Video S1).
Briefly, HeLa cells were treated with TPE-4EP+ as the PS and
subsequently stained with annexin V and PI, followed by light
exposing to induce cell death. As shown in Figure 3, TPE-4EP+
anchored to mitochondria of the healthy cells at the beginning.
After continuous 5-minutes laser irradiation, fluorescent signal
from FITC emerged surrounding the cell membrane while PI
signal was still invisible. As annexin V is able to stain early-stage
apoptotic cells while PI only stains dead cells or late-stage
apoptotic cells, the result of 5-minute laser irradiation indicates
that the cells were at the early stage of apoptosis.37 With
continuous irradiation, the integrity of the cell membranes was
destroyed and the PI signal became obvious. It is worth noting
that TPE-4EP+ underwent fluorescence translocation from
mitochondria to nucleus gradually and the signal mainly located
in nucleus at final point. Meanwhile, cell apoptosis under light
Figure 2. Confocal imaging of HeLa Cells (A−D) before (0 min,
upper panel) and (E−H) after white light irradiation for 10 min
(lower panel) stained with (A,E) TPE-4EP+, (B,F) PI and (C,G)
Bright-field. (D, H) Merged images of Panel (A) and (B), (E) and
(F).
The unique emission enhancement phenomenon, good water-
solubility and intense long-wavelength emission of these
molecules encouraged us to investigate its bioapplication.31,32
Figure S6 illustrated the colocalization image of stained cell.
Clearly, the mitochondria can be visualized with bright
fluorescence and showed high specificity (Figure S7). Their
mitochondria-targeting behavior could be attributed to
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irradiation was confirmed by annexin V-FITC/PI staining through
during PDT treatment may be correlated with the specific
interactions between molecules and some biomolecules in
nucleus.
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flow cytometry (Figure S17), which was in agreement with that of
cell-imaging experiments. Collectively, these evidences indicated
that mitochondria-to-nucleus translocation of TPE-4EP+ was
induced by cell apoptosis, which is the primary cause of effective
therapeutic process. More importantly, the different stage of
apoptosis could be differentiated clearly using TPE-4EP+.
DNA, one of the most abundant components in nucleus, was
reported previously to exhibit high affinity for cationic AIEgens
by electrostatic force.23,40,41 To testify the principle of the nucleus-
targeting, we used calf thymus DNA (ctDNA) as a model (Figure
4E, Figure S24). Three molecules exhibited the PL enhancing
effect after adding ctDNA in the order of TPE-2EP+ ＜ TPE-
3EP+ ＜ TPE-4EP+, which was in accordance with the sequence
of increasing numbers of charge carried. This interesting result
indicates that the molecules with more positively charged
pyridinium moieties on their branch could dock on the surfaces
and in the cavities of the negatively charged DNA via electrostatic
interactions. The intramolecular motions of phenyl rings are thus
restricted to hinder nonradiative decay and strong emission is
activated.40
Positively charged pyridinium salt in molecules has been
reported to be an important functional group processing efficient
PDT effect.31,35 Therefore, the phenomenon of apoptosis process
under light irradiation prompted us to investigate the
phototoxicities of the molecules. First, the extracellular
experiment was conducted by evaluating the efficiency of 1O2
generation preliminarily, which plays a key role in PDT.38,39
Under 80s of light irradiation, over 80% of 9,10-
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anthracenediylbis(methylene)dimalonic
acid
(ABDA),
a
commercially available 1O2 indicator , was consumed at the
presence of TPE-4EP+ in water solution. This approximated to a
decomposition rate of 118.5 nmol min-1, while that of TPE-2EP+
and TPE-3EP+ was 48.1 and 69.5 nmol min-1, respectively
(Figure S18). All the molecules are more effective than the
commercial photosensitizer Rose Bengal (16.7 mmol min-1).
Then, we further studied the PDT effect of molecules as PSs by
quantitatively evaluation on HeLa cells through MTT assay.
Under white-light irradiation, three molecules exhibited superior
phototoxicity (Figure S19). Meanwhile, the bright-green emission
of dichlorofluorescein diacetate (DCFDA, a reactive oxygen
species indicator) from cell incubation experiments also verified
the 1O2 production (Figure S20). Remarkably, the molecules
exhibited high cell viability in dark condition. Low cytotoxicity
towards HLF cells was also observed, suggesting their
biocompatibility (Figure S21). Based on the results, 1O2
generation efficiency is directly correlated with the charge
numbers of molecules, in the sequence of TPE-2EP+ ＜ TPE-
3EP+ ＜ TPE-4EP+. Collectively, without other extra agents,
real-time in situ monitoring of apoptosis during PDT is afforded
by simply visualizing the translocation phenomenon of TPE-4EP+
in cells, making it a self-reporting photodynamic theranostic
system.
Based on the above studies, we proposed the mechanism of
mitochondria-to-nucleus translocation of TPE-4EP+ induced by
1O2 generation upon PDT treatment (Figure 1B). At the
beginning, TPE-4EP+ stains mitochondria specifically due to its
high affinity to negatively charged mitochondrial membrane.
After laser or white light irradiation, photosensitized 1O2
generates and induces the cell apoptosis, which involves the
depolarization of the mitochondrial membrane potential, increase
of cell permeability, and degradation of nuclear DNA. Therefore,
TPE-4EP+ could detach and lose its targeting ability after
mitochondrial dysfunction during cell death by photodynamic
effect, subsequently relocate and illumine the nucleus by binding
to the nuclear DNA strongly through electrostatic interaction.
In summary, TPE-4EP+ can not only induce cell apoptosis by
1
the intrinsic high O2 generation efficiency under irradiation, but
also clearly differentiate health and apoptotic cells without any
additional agents. Since TPE-4EP+ specifically stains HeLa
cancer cells, cytotoxicity to normal cells can be mitigated.
Particularly, the fluorescent PS can serve as a self-reporter to
monitor the PDT process, such as where the cancer cell locates,
how the PDT performs, and when is the end point of PDT. Our
work opens a new way for visualizing the photodynamic therapy
so that the control the phototoxicity dose is possible. In addition,
the working principle of TPE-4EP can provide strategy for the
rational design of new generation of PS.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Detailed methods, characterizations and supplementary figures
(PDF)
Movie of real-time dynamic change of TPE-4EP+ (avi.)
Figure 4. Confocal images of Fixed HeLa cells stained with 1 μM
of (A)TPE-4EP+, 200 nM of (B) MTO and 1 μM of (C) DAPI.
(D) Merged images of panels (A), (B) and (C). (E) The Plot of
elevated PL intensity at 605 nm versus the ctDNA. Inset:
photographs of TPE-4EP+ in water solution in the absence and
presence of ctDNA taken under 365 nm UV irradiation.
AUTHOR INFORMATION
Corresponding Author
*tangbenz@ust.hk
Author Contributions
§T.Z. and Y.L. contributed equally to this work.
To decipher the rationale behind the translocation of TPE-4EP+
during PDT treatment, 4% paraformaldehyde-fixed HeLa cells
were treated with three molecules. The result showed that TPE-
2EP+ can still stained the mitochondria of fixed cell while TPE-
3EP+ spread throughout the whole cells and lost the specificity
(Figure S22–S23). However, for the cell imaging of TPE-4EP+,
only nucleus showed fluorescence and high specificity (Figure
4A–D). It is proposed that the different fluorescent performance
Notes
The authors declare the following competing financial interest(s):
T.Z., Y.L., Z.Z. and B.Z.T. are the inventors of a patent filed by
Hong Kong University of Science and Technology.
ACKNOWLEDGMENT
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Differentiating the Early and Late Stages of Apoptosis Mediated by H2O2.
J. Mater. Chem. B 2016, 4 (33), 5510–5514.
This work was financially supported by the National Science
Foundation of China (21788102), the Research Grants Council of
Hong Kong (16305518, C6009-17G and A-HKUST605/16), the
Innovation and Technology Commission (ITC-CNERC14SC01
and ITS/254/17), the National Key Research and Development
program of China (2018YFE0190200) and the Science and
Technology Plan of Shen-zhen (JCYJ20160229205601482 and
JCYJ20170818113602462).
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