A triazole-based fluorescence probe for detecting Hg2+ ions and its biological application

Article abstract of DOI:10.1002/bio.3705

A new compound, ethyl 5-phenyl-2-(p-tolyl)-2H-1,2,3-triazole-4-carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N-dimethylformamide–H₂O (pH?7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open-ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization-mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.

Full text of DOI:10.1002/bio.3705

Research Article
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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 15426−15435
Nitroreductase-Activatable Theranostic Molecules with High PDT
Efficiency under Mild Hypoxia Based on a TADF Fluorescein
Derivative
Zhiwei Liu,† Fengling Song,*,†,§ Wenbo Shi,‡ Gagik Gurzadyan,† Huiyi Yin,† Bo Song,‡ Ri Liang,†
and Xiaojun Peng†
‡
†State Key Laboratory of Fine Chemicals and School of Chemistry, Dalian University of Technology, 2 Linggong Road, Dalian
116024, P. R. China
§Institute of Molecular Sciences and Engineering, Shandong University, Qingdao 266237, P. R. China
S
* Supporting Information
ABSTRACT: High specificity detection and site-specific
therapy are still the main challenges for theranostic anticancer
prodrugs. In this work, we reported two smart activatable
theranostic molecules based on a thermally activated delayed
fluorescence fluorescein derivative. Nitroreductase induced by
a mild hypoxia microenvironment of a solid tumor was used to
activate the fluorescence and photodynamic therapy (PDT)
efficiency by employing the intramolecular photoinduced
electron transfer mechanism. A high PDT efficiency under
10% oxygen concentration was achieved, which is better than
that of porphyrin (PpIX), a traditional photosensitizer. Such
an excellent PDT efficiency can be attributed to lysosome
disruption because the theranostic molecule can specifically enter the lysosomes of cells. Importantly, the strategy of targeting
the mild hypoxic cells in the edge of tumor tissue could heal the “Achilles’ heel” of traditional PDT. We believe that this
theranostic molecule has a high potential to be applied in clinical investigation as a theranostic anticancer prodrug.
KEYWORDS: photodynamic therapy, hypoxia, cancer theranostics, thermally activated delayed fluorescence, nitroreductase,
photoinduced electron transfer
Hypoxia is an important feature of the tumor microenviron-
ment due to insufficient oxygen supply in rapid proliferation of
cancer cells.14−16 It seems a smart strategy to activate PDT
efficiency by exploiting hypoxia as the activating factor for
PDT. However, tumor hypoxia is also known as the “Achilles’
heel” of traditional PDT17,18 because severe tumor hypoxia
hampers therapeutic outcomes of oxygen-dependent PDT and
PDT potentiates hypoxia. Recently, Hanaoka et al. reported a
mild hypoxia-activatable photosensitizer for PDT based on a
selenorosamine dye.19 Under 5% oxygen concentration, the
reported smart photosensitizer can be activated by azor-
eductase and give a high PDT efficiency. This work opens a
new window to resolve the notorious dilemma between
efficient PDT and the hypoxia microenvironment. Cancer cells
under mild hypoxia are considered as important targets
because stemness properties of cancer cells are directly related
to the mild hypoxia microenvironment.20 Meanwhile, the
margin of the solid tumor is reasonably the ideal target for
PDT, because the gradiently decreased oxygen concentration
INTRODUCTION
■
Theranostic anticancer prodrugs have caused extensive
concern in the last decade because they can diagnose and
treat the nidus region at the same time.1−6 Traditional
theranostic prodrugs are constituted by a fluorophore moiety
for a diagnosis response signal and a chemotherapy prodrug
moiety for treatment. This combination has some problems,
such as a complicated structure that is difficult to be
synthesized, an unpredictable effect of the prodrug structure
when linked to the fluorophore, and the aporia of the drug site-
specific release. Among them, the lack of the drug site-specific
release will lead to irreversible damage to normal tissue that
limits their practical application. Photodynamic therapy (PDT)
is a promising solution for site-specific treatment by selective
irradiation of tumor regions.7,8 However, the margin of tumor
regions is usually not clear, the shape of the tumor is irregular,
and the scattered light of irradiation is difficult to be
controlled. It is not easy to direct light only to microscopic
tumors that are surrounded by healthy tissue that is inevitably
injured. So, it is very desirable to have “smart” activatable PDT
theranostic molecules to differentiate the nidus region from the
surrounding healthy tissues and site-specifically kill tumor
cells.9−13
Received: March 12, 2019
Accepted: April 4, 2019
Published: April 4, 2019
© 2019 American Chemical Society
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Scheme 1. (a) The Design Strategy for the Smart Theranostic Molecule; (b) the Molecular Structure of Compounds 1−3
from outside to inside of the solid tumor makes the outer edge
of the tumor to become the mild hypoxia area, and good PDT
efficacy can diminish the tumor volume to make a new margin
in turn. By this strategy, the severe hypoxic area inside the
tumor will be continuously becoming mild hypoxic, which will
be very favorable for improving the efficiency of PDT. The
Achilles’ heel of traditional PDT could be healed. So, it is
desirable to develop activatable PDT photosensitizers targeting
the margin of a solid tumor. However, to the best of our
knowledge, such smart PDT photosensitizers working in mild
hypoxia microenvironment are very few.
Photosensitizers with thermally activated delayed fluores-
cence (TADF) are the emerging triplet excited-state
chromophores without the introduction of heavy atoms or
transition metals. TADF compounds have attracted wide
attention recently for their breakthrough potential in the
organic light-emitting diode (OLED) field.21−23 However,
these TADF compounds are usually hydrophobic and are not
able to be directly used in biological and medical fields.24 In
our previous work, we developed a new class of TADF
photosensitizers based on fluorescein derivatives. These TADF
dyes show low-toxicity and were used as chemosensors for
cysteine or hypochlorite in living cells.25,26
There are important advantages in using our TADF
fluorescein derivatives as theranostic molecular probes.
Normally, PDT photosensitizers have a strong intersystem
crossing (ISC) process to form a triplet excited state.
Meanwhile, they cannot keep emitting strong fluorescence or
phosphorescence at room temperature. However, our fluo-
rescein derivatives have a narrow energy gap between the
singlet excited state (S1) and the triplet excited state (T1). So, a
delayed fluorescence can be easily observed because of a quick
intersystem crossing (ISC) process and a quick reverse ISC
process between the two excited states. In other words, these
TADF dyes contain steady S1 and good fluorescence
properties. The energy from the triplet excited state can be
used for PDT by sensitization of oxygen, and the fluorescence
from the singlet excited state can be used for the optical
sensing signal. On the other hand, our TADF fluorescein
derivatives have been found to be lysosome targeted, so the
singlet oxygen produced in situ on lysosomes could induce
lysosome disruption to achieve a better PDT effect. These
characters provide them a potential to become better
theranostic molecular probes than other PDT photosensitizers.
Nitroreductase (NTR) is known as the most studied for the
overexpression of reductases inside tumor tissue induced by
hypoxia. In this work, we try to develop smart PDT
photosensitizers activated by NTR with our low-toxic TADF
fluorescein derivatives. This smart activation strategy is based
on the photoinduced electron transfer (PET) mechanism that
has often been employed in designing fluorescence probes for
NTR.27−31 The PET process in this work is expected to cage
not only the fluorescence decay of the singlet excited state but
also the PDT efficiency in which PET competes with the ISC
process and weakens the 1O2 production (Scheme 1a).
Importantly, mild hypoxia up to 10% oxygen concentration
has been found to induce enough NTR to switch on the
fluorescence of many probes.29,32−34 Herein, the PDT
efficiency is expected to be activated accompanying with the
disappearance of the PET process when an enzymatic cleavage
reaction occurs, which is triggered by NTR induced under mild
hypoxia.
EXPERIMENTAL PROCEDURES
■
General Information. Absorption spectra were measured using a
UV−vis spectrophotometer (Agilent Tech). Fluorescence spectros-
copy was performed on a fluorometer (Agilent Tech). Femtosecond
transient absorption spectra were measured by a home-made ultrafast
pump−probe setup.35 The pulse duration was 30 fs. Nanosecond
fluorescence lifetimes were determined using the TCSPC technique
on the HORIBA Jobin Yvon IBN photo counting fluorescence system
with NanoLED excitation at 456 nm. Microsecond fluorescence
lifetimes were determined using the HORIBA Jobin Yvon IBN photo
counting fluorescence system with spectra-LED excitation at 452 nm.
Nanosecond time-resolved transient absorption spectra were obtained
using an LP920 laser flash photolysis spectrometer (Edinburgh
Instruments, U.K.). Fluorescence imaging was performed using an
Olympus FV-1000 inverted fluorescence microscope with a 60×
objective lens. The time-resolved fluorescence imaging was performed
on a laboratory-use true color time-resolved luminescence micro-
scope.
Methods for Fluorescence Spectroscopy. The stock solutions
of compounds were prepared in dimethyl sulfoxide and stored at 4 °C
in a refrigerator. NTR was purchased from Sigma-Aldrich in powder.
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The stock solution of NTR was prepared in phosphate-buffered saline
(PBS) buffer (pH 7.4) and preserved in small batches at −20 °C in
the refrigerator. NADH was purchased from J&K Scientific Company
in powder. The NADH was prepared when it was needed.
Compound 1 or 2 (30 μL, 10 μM) and NADH (30 μL, 50 μM)
were added into pH 7.4 PBS buffer (210 μL). To the above mixture,
NTR (30 μL) was added. The resulting mixture was shaken for 1 s
and incubated at 37 °C for a certain time. Before fluorescence
analysis, CH3CN (2.7 mL) was added to stop enzyme catalysis.
High-Performance Liquid Chromatography (HPLC) Experi-
ment. HPLC was carried out with H2O (0.3% HAc and 0.3% TEA)
and MeOH as eluents by C18 reverse phase chromatography.
Gradient elution conditions: H2O/MeOH = 30:70 in the first 20 min,
then 0:100 in the next 30 min.
nm and collected at 500−525 nm. The red channel was excited at 488
nm and collected at 610−660 nm.
Lysosome Disruption Assay. The HeLa cells were preincubated
under normoxia or hypoxia conditions (oxygen concentrations 10%)
for 6 h, then incubated with compound 2 (10 μM) for 90 min at the
same conditions, the medium was replaced with fresh medium and
balanced in normoxia or hypoxia conditions for another 60 min. After
that, the cells were subjected to photoirradiation with 590 nm LED
(16 mW/cm2) under the same conditions as before for 20 min. After
photoirradiation, the cells were stained with acridine orange (AO, 5
μM) for 20 min. The green channel was excited at 488 nm and
collected at 515−545 nm. The red channel was excited at 488 nm and
collected at 610−640 nm.
Confocal Microscopy Imaging of Cells with AM Staining.
The HeLa cells were preincubated under normoxia or hypoxia
conditions (oxygen concentrations 10%) for 6 h, then incubated with
compound 2 (10 μM) for 90 min at the same conditions, the medium
was replaced with fresh medium and balanced in normoxia or hypoxia
conditions for another 60 min. After that, the cells were subjected to
photoirradiation with 590 nm LED (16 mW/cm2) under the same
conditions as before for 20 min. After photoirradiation, the cells were
assessed using a LIVE/DEAD cell viability assay by staining with
calcein AM (2 μM) for 30 min. The channel of AM was excited at 488
nm and collected at 500−550 nm.
Cell Viability with Photoirradiation. The cells were seeded at a
density of 1 × 105 cells/mL in a 96-well plate for 24 h at 5% CO2
atmosphere, then the cells were incubated for another 6 h under
normoxia (oxygen concentrations 21%) or hypoxia (oxygen
concentrations 10% or 1%) at 37 °C. After this process, the cells
were incubated with different concentrations of compound 2,
compound 3, or PpIX for 90 min under normoxia or hypoxia
conditions. Then the medium was removed, and the fresh medium
was added, and the plate was balanced in normoxia or hypoxia
conditions for another 60 min. After that, the cells were irradiated by
a 590 nm LED lamp (16 mW/cm2) under the same conditions as
before for 20 min. The cell viability was measured through 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as-
says.
FACS Analysis of Annexin V-FITC and PI-Labeled HeLa
Cells. The HeLa cells were incubated under normoxia or hypoxia
conditions (oxygen concentrations 10%) for 6 h and then incubated
with compound 2 (10 μM) for 90 min at the same conditions, the
medium was replaced with fresh medium and balanced in normoxia or
hypoxia conditions for another 60 min. The cells were subjected to
photoirradiation with 590 nm LED (16 mW/cm2) under the same
conditions as before for 20 min. After photoirradiation, the cells were
incubated with FITC (5 μL, KeyGEN BioTECH, Nanjing, China)
and PI (5 μL, KeyGEN BioTECH, Nanjing, China) for 30 min. FACS
analysis was performed to quantify the extent of apoptosis on an
Attune NxT Flow Cytometer (Thermo Fisher Scientific).
Docking Calculations. The binding affinity calculations between
the compounds and NTR were carried out on AutoDock software
(4.2.6 version). The PDB code of NTR was 4DN2. The docking
results and figures were obtained from AutoDock and LigPlot +.
Singlet Oxygen Trap Experiments. 1,3-Diphenylisobenzofuran
(DPBF) was used directly after purchase, in a typical procedure for
the detection of singlet oxygen generation, a photosensitizer (10 μM)
and DPBF (O.D ∼ 1.0) were mixed in CH3CN. Photoirradiation was
performed using 590 nm LED (16 mW/cm2). The distance from the
samples and the light was 5 cm. During the entire measurement, the
sample needs to be stirred all the time to ensure that the samples were
homogenized before absorbance measurements. The absorbance
decrease of DPBF at 410 nm was monitored after photoirradiation.
Cell and Culture Conditions. HeLa cell line was purchased from
the Chenyu biological company (Dalian, China). The culture medium
of HeLa cells was Dulbecco’s modified Eagle medium (Kaiji, Nanjing,
China) containing 10% fetal bovine serum (Sijiqing, Zhejiang,
China). The culture conditions of HeLa cells were 37 °C in an
atmosphere of 5% CO2 in an incubator. HeLa cells were seeded in a
20 mm glass bottom dish from NEST company and incubated for 24
h. Anaero Pack-Anaero and Anaero Pack-Micro Aero (Mitsubishi Gas
Chemical Company, Japan) were used to create hypoxia conditions (1
and 10% oxygen levels).
Confocal Fluorescence Imaging of Cells. The HeLa cells were
seeded in a glass bottom dish and incubated for 24 h under normoxia
conditions (oxygen concentrations 21%). Then the cells were
incubated for another 6 h at 37 °C under normoxia or hypoxia
(oxygen concentrations 10 and 1%). Under a normoxia or hypoxia
atmosphere, compound 2 (10 μM) was added and incubated for
another 90 min. The inhibition experiment was performed by
preincubating the cells with dicoumarin (0.2 mM) for 30 min and
then incubating with compound 2 for another 90 min under hypoxia
(1% O2). The cells were washed with PBS buffer (pH = 7.4) three
times before imaging. Fluorescence images were collected at 610−660
nm upon excitation at 488 nm.
Intracellular Singlet Oxygen Detection. 2,7-Dichlorofluores-
1
cein diacetate (DCFH-DA) was used to prove the generation of O2
Luminescence Imaging of Mice with Endogenous NTR. The
luminescence imaging of HeLa tumor mice (n = 4, weight ∼ 25 g)
was carried out on a living small animal luminescent imaging analysis
system NightOWL II LB983 from Berthold company of Germany.
The SCID/BALB/c-nude mice were purchased from Dalian Medical
University (Dalian, China, Ethics certificate number is 2018-043).
The HeLa cell lines (2 × 107 cells/mL, 0.1 mL) were implanted by
subcutaneous injection into the oxter of nude mice to establish the
HeLa tumor model. The tumor-bearing mice were feed for 20 days
when a 1.0 cm diameter tumor model was formed. One group of
anesthetic mice was injected intratumorally with compound 2 (0.1
mM, 100 μL), the other mouse was injected intratumorally with
dicoumarin (0.2 mM, 50 μL) for 30 min before the intratumoral
injection of compound 2 (0.1 mM, 100 μL). The time-dependent
fluorescence changes were observed at every 5 min within 80 min
after injection. Then, all mice were sacrificed and the tumors were
taken out and embedded with embedding medium. The slices with a
thickness of 50 μm were obtained by cryosection at −20 °C with a
microtome CM1860 UV from Leica Company of Germany.
in HeLa cells. The HeLa cells were preincubated under normoxia or
hypoxia conditions (oxygen concentrations 10 and 1%) for 6 h, then
incubated with compound 2 (10 μM) for 90 min at the same
conditions, the medium was replaced with fresh medium and balanced
in normoxia or hypoxia conditions for another 60 min. After that, the
cells were subjected to photoirradiation with 590 nm LED (16 mW/
cm2) under the same conditions as before for 10 min. After
photoirradiation, the cells were stained with DCFH-DA (10 μM) for
20 min. The green channel was excited at 488 nm and collected at
500−520 nm.
Subcellular Colocalization Assay of Cells. The HeLa cells were
seeded in a glass bottom dish and incubated for 24 h under normoxia
conditions (oxygen concentrations 21%). The cells were incubated for
another 6 h at 37 °C under hypoxia (oxygen concentrations 1%). The
subcellular colocalization assay was performed under a hypoxia
atmosphere. In the beginning, compound 2 (10 μM) was added and
the cells were incubated for 90 min. After the culture medium was
removed, lysosome green (1 μM) was added, and the cells were
incubated for another 30 min. The green channel was excited at 488
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Photocytotoxic Effect of Tumor-Bearing Mice. The HeLa
tumor-bearing mice were chosen for this research, the HeLa cell lines
(2×107 cells/mL, 0.1 mL) were implanted by subcutaneous injection
into the oxter of nude mice to establish the HeLa tumor model. The
phototoxicity tests were conducted when the tumor volume was close
to 50 mm3, 16 mice were randomly divided into four groups, the first
group of mice was intratumorally injected with saline (100 μL)
without irradiation (G1), the second group of mice was only
irradiated with 590 nm LED (16 mW/cm2) for 30 min on the tumor
region (G2), the third group was intratumorally injected with
compound 2 (0.1 mM, 100 μL) without irradiation (G3), the last
group of mice was intratumorally injected with compound 2 (0.1 mM,
100 μL) at first and then irradiated with 590 nm LED (16 mW/cm2)
for 30 min at the tumor region for 40 min after injection (G4). All the
mice were treated once every three days on the “0 day”, “3 day”, “6
day” and “9 day”. No treatment was done for the last 6 days. The
tumor volume and body weights were measured every 3 days during
the treatment. The tumor volumes were calculated using the formula
V = (a × b2)/2 (a is the longest diameter of tumors, b is the shortest
diameter of tumors, and a, b was measured by a caliper). When the
tumor sizes of the G1 exceeded 800 mm3, the measurements of all
groups were stopped and the mice were sacrificed.
Hematoxylin and Eosin (H&E) Staining. The tumors of the
four groups of mice were fixed with 10% formaldehyde solution and
then embedded with paraffin to slice into 5 μm sections. These
sections were stained with hematoxylin and eosin (H&E) using
standard methods and observed under a microscope.
Figure 1. (a) Normalized absorption and (b) fluorescence emission
spectra of compounds 1 (10 μM), 2 (10 μM), and 3 (10 μM) in PBS
buffer (λex = 488 nm). (c, d) Dynamic changes of fluorescence
intensity were recorded for compound 1 and compound 2 response to
NTR with different response times. (e, f) The selectivity of
fluorescence responses of compound 1 and compound 2 to different
kinds of species (1) control, (2) NaCl (50 mM), (3) KCl (50 mM),
(4) CaCl2 (50 mM), (5) MgCl2 (50 mM), (6) vitamin C (1 mM),
(7) Lys (1 mM), (8) Ser (1 mM), (9) Arg (1 mM), (10) Leu (1
mM), (11) Asp (1 mM), (12) Gly (1 mM), (13) Tyr (1 mM), (14)
GSH (10 mM), (15) Hcy (1 mM), (16) Cys (1 mM), (17) KO2 (1
mM), (18) glutathione reductase (GR, 10 μg/mL), (19) leucine
dehydrogenase (LEDH, 10 μg/mL), (20) formate dehydrogenase
(FDH, 10 μg/mL), (21) DT-diaphorase (5 μg/mL), (22) BSA (1
mg/mL), (23) HSA (1 mg/mL), (24) CES1B (100 μg/mL), (25)
CES1C (100 μg/mL), (26) CES2 (100 μg/mL), (27) NTR (4 μg/
mL) in PBS buffer at 37 °C in the presence of probes (10 μM) and
NADH (50 μM). The fluorescence intensities were recorded at 635
nm with a 15 min response time after the NTR was added to the
mixture (λex = 488 nm).
RESULTS AND DISCUSSION
■
Design and Synthesis of Compound 1 and Com-
pound 2. Alkylation of the hydroxyl of fluorescein can easily
tune the fluorescence properties due to the formation of a
spiro-lactone structure.36−39 Such a smart theranostic molecule
1 was first designed by the above strategy and synthesized from
our reported TADF compound 3 (Scheme 1b).40 As expected,
compound 1 was synthesized by the introduction of the 4-
nitrophenylchloroformate into the hydroxy of compound 3
(Scheme S1). Meanwhile, a by-product was obtained at the
same time, which was further confirmed to be compound 2 by
1
MS and H, 13C NMR, 2D-HMBC. Then compound 2 was
resynthesized by a straight way in which compound 3 was
treated with 4-nitrobenzyl bromide. Both compound 1 and
compound 2 were checked as the smart activatable theranostic
molecules in the following NTR response experiments. We
also call compounds 1−2 as probes.
Spectroscopic Responses of Compounds 1−2 to NTR.
In the beginning, the responses of the probes to NTR were
tested in aqueous solutions. Both the two probes are in the
“off” state when compared with strong fluorescence of
compound 3 according to their absorption spectra shown in
Figure 1a. Their turn-on response toward NTR was
investigated by fluorescence spectra (Figure 1b). The sensing
mechanisms of these two compounds were proposed as shown
in Scheme S2, based on previously reported works.34,41,42 The
sensing mechanisms were verified by HPLC (Figures S1 and
S2) and MS (Figures S3 and S4). During the turn-on process,
both compounds 1 and 2 can be transferred into compound 3.
Unexpectedly, probe 2 exhibits a much faster response and
better selectivity than probe 1 (Figure 1c−f). Theoretical
calculations were employed to clarify why compound 2 is the
better hypoxia-activatable theranostic molecular probe than
compound 1. Docking calculations were carried out for the
interaction of compound 1 and compound 2 with NTR.
Although both 1 and 2 form one hydrogen bonding with NTR
(Figure S5), their docking affinities of two compounds have an
obvious difference. From Figure S6, the docking energy of
compound 1 with NTR is −2.58 to −8.62 kcal/mol, whereas
the docking energy of compound 2 is −3.98 to −11.36 kcal/
mol. The lower binding energy denotes the higher affinity
between compound 2 and NTR.34
After probe 2 was selected as the better photosensitizer for
the enzyme activation process, its enzyme catalysis properties
were evaluated in detail. The optimum conditions for this
enzyme catalysis reaction were found to be 37 °C and pH 7.4
(Figure S7). The time-dependent sensing process began to
level off after 5 min, which means the activation can be
completed very fast (Figure S8). For the sake of accuracy, the
15 min sensing time was chosen to achieve the working curve
for determination of a concentration range of 0−0.75 μg/mL
of NTR (Figure S9). In the chosen sensing conditions, the
working curve shows a good linear (R = 0.996), and the
detection limit was deduced to be 8 ng/mL.
PDT Activation Mechanism of Compound 2. Since the
PET mechanism is expected to cage the fluorescence and the
PDT efficiency of the smart theranostic molecule, we move on
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compound 2 and compound 3 (600 nm kinetics) decay
biexponentially. The fast process (32 ps for compound 2, 6 ps
for compound 3) is assigned to the vibrational cooling,
whereas the longer decay (690 ps for compound 2, 1270 ps for
compound 3) corresponds to the S1 state lifetime. This result
is consistent with fluorescence lifetime results of compound 2
(0.56 ns) and compound 3 (1.73 ns) (Figure S11 and Table
S2). The shorter S1 lifetime of compound 2 compared with
compound 3 is due to the competition of PET with the
fluorescence decay process. These results also explain the
stronger fluorescence intensity of compound 3 compared with
compound 2, which make compound 2 a good turn-on
fluorescence probe for NTR to meet the objective of diagnosis.
To make compound 2 become a good PDT photosensitizer to
fulfill the goal of treatment, its T1 lifetime should also be
triggered by the PET process. As shown in Figures 2c,d and
S12 and Table S3 (nanosecond transient absorption data),
both compound 2 and compound 3 have a long-lived flat
transient absorption appearing in the 450−650 nm region, but
the triplet state lifetime of compound 2 (1.74 μs) is much
shorter than compound 3 (16.3 μs). It means that the PET
process in compound 2 does complete with not only the decay
of S1 but also the decay of T1. Therefore, the longer T1 of
compound 3 than that of compound 2 will result in efficient
activation of the PDT efficiency.
Figure 2. (a, b) Femtosecond transient absorption spectra of
compound 2 (500 μM) and 3 (100 μM) in deaerated ethanol at
different decay times (λex = 450 nm). (c, d) Nanosecond transient
absorption spectra of compound 2 (10 μM) and 3 (10 μM) in
deaerated ethanol at different decay times (λex = 532 nm).
After that, the PDT abilities of compounds 2 and 3 were
checked whether singlet oxygen can be efficiently generated by
the two compounds under photoirradiation. We used 1,3-
diphenylisobenzofuran (DPBF) as a singlet oxygen trap to
check the ability of production singlet oxygen of compound 2
and compound 3 in acetonitrile.43−45 As shown in Figure S13,
the rate of singlet oxygen generation by compound 3 was much
faster than by compound 2. This result indicates that the PDT
performance of compound 2 could be efficiently activated
1
according to the different abilities of O2 production between
compounds 2 and 3.
Fluorescence and PDT Activation of Compound 2 in
Living Cells. Since the response of compound 2 to NTR has
been well investigated in aqueous solutions mainly containing
PBS buffer, we try to assess the response of compound 2 to
NTR in living HeLa cells by one-photon and two-photon
confocal fluorescence imaging. In the one-photon fluorescence
imaging experiments, the HeLa cells were incubated under
three different oxygen concentrations (Figure 3a−c). With the
decrease of the O2 concentration from 21 to 1%, the
fluorescence intensity of the tumor cells was increased
correspondingly. The intracellular inhibition experiments
with dicoumarin, a known inhibitor,34,46,47 support the fact
that the enhanced fluorescence was due to the endogenous
NTR (Figure 3d). The two-photon imaging data further
support our above explanation (Figure S14 and S15) and offer
compound 2 a two-photon PDT potential for deeper tissue
treatment.7,8,48,49 These results prove that the endogenous
NTR can catalyze the enzymatic cleavage reaction of
compound 2 to compound 3 in living tumor cells.
Furthermore, the longer T1 activation process can be visually
captured by time-resolved luminescence imaging. According to
our previous work, the long-living triplet excited state in our
fluorescein derivatives can bring out the long TADF lifetime by
the RISC process.25,40 The lifetime of compound 3 is 22.14 μs
in deaerated ethanol, much longer than the luminescence
lifetime of compound 2 (Figure S16 and Table S4). As shown
in Figure 3e, the activated long-lived luminescence was
Figure 3. (a−c) Confocal fluorescence images of HeLa cells
incubated with compound 2 (10 μM) for 90 min under different
oxygen concentration conditions ((a) 21%; (b) 10%; (c) 1%). (d)
Confocal fluorescence images of inhibition experiments with
dicoumarin (HeLa cells were preincubated with dicoumarin (0.2
mM) for 30 min, then incubated with compound 2 (10 μM) for 90
min under 1% oxygen concentration). (e) Time-resolved fluorescence
images of HeLa cells incubated with compound 2 (10 μM) for 90 min
under 1% oxygen concentration. The scale is 30 μm.
to clarify how the PET process affects the S1 and T1 states of
compound 2. We obtained femtosecond and nanosecond
transient absorption spectra for compound 2 and compound 3.
As shown in Figures 2a,b and S10 and Table S1, S1 states of
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Figure 4. (a, b) Cell viabilities of HeLa cells based on the MTT assay of PpIX and compound 2 under 590 nm LED light irradiation. (c) AM
staining of compound 2-treated HeLa cells without (c1, c3) or with (c2, c4) irradiation in normoxia (c1, c2) or hypoxia (10%, c3 and c4)
conditions. (d) FACS of annexin V-FITC and PI-labeled HeLa cells without (d1, d3) or with (d2, d4) irradiation in normoxia (d1, d2) or hypoxia
(10%, d3, d4) conditions. Scale is 30 μm.
captured successfully by time-resolved luminescence imaging
experiments (delayed time 33 μs) accompanying the enzymatic
cleavage reaction of compound 2 to compound 3 under
hypoxia conditions.
Then the NTR-activated PDT efficiency was checked in
living tumor HeLa cells by MTT assay under three different
oxygen concentrations. As shown in Figure 4a, the PDT
Figure 5. Subcellular colocalization assay of HeLa cells. Cells were
incubated with compound 2 (10 μM) for 90 min, then incubated with
efficiency of the traditional photosensitizer PpIX is highly
lysosome green (1 μM) for 30 min. (a1) Image of lysosome green;
dependent on oxygen concentration. Server hypoxia at 1%
(a2) the image of compound 2; (a3) the merged images of a1, a2, and
oxygen concentration will dramatically hamper the PDT
bright field; (a4) the correlation plot of lysosome green and
efficiency. Interestingly, the efficiency of PDT at 10% oxygen
concentration remains similar to that at ambient oxygen
concentration, which is consistent with others’ work.50 The
similar results were achieved for compound 3 (Figure S17). It
means that the PDT performance of PpIX and compound 3
maintain almost the same level of photocytotoxicity between
10 and 21% oxygen concentration. However, the PDT
efficiency of compound 2 at 10% oxygen concentration is
much higher than at 21% (Figure 4b), and the half-maximal
inhibitory concentration of compound 2 (IC50 = 5.91 μM) was
even lower than that of PpIX (IC50 = 6.46 μM) at 10% oxygen
concentration, which means our activation strategy is
successful.
compound 2.
The success of the PDT activation process should attribute
that mild hypoxia at 10% oxygen concentration can induce
enough NTR to trigger the transfer from compound 2 to 3. It
is noteworthy in the confocal fluorescence imaging experiment
that mild hypoxia conditions (10% oxygen concentration)
were found to be able to induce enough NTR to switch on the
fluorescence (Figure 3b). It means the endogenous NTR can
catalyze the enzymatic cleavage reaction of compound 2 to
compound 3 in living tumor cells even under 10% oxygen
Figure 6. Acridine orange (AO) staining confocal fluorescence images
of HeLa cells incubated with compound 2 (10 μM) for 90 min. (a1)
HeLa cells were treated under normoxia conditions without
irradiation; (a2) HeLa cells were treated under normoxia conditions
with irradiation; (a3) HeLa cells were treated under hypoxia
conditions without irradiation; (a4) HeLa cells were treated under
hypoxia conditions with irradiation; (b1−b4) the bright field images
correspond to (a1)−(a4). Scale is 30 μm.
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Figure 7. Fluorescence imaging of endogenous NTR activities in HeLa tumor-based SCID/BALB/c-nude mice by intratumor injection with
compound 2 (0.1 mM, 100 μL). (a) Time-dependent fluorescence imaging of the mouse tumor model. (b) Time-dependent fluorescence
quantification of the tumor sections in (a). Error bars indicate standard deviation (SD) (n = 2).
concentration, which leads to efficient PDT under mild
hypoxia conditions.
According to our previous work,40 compound 3 was found to
localize in the lysosome. These results prove that the
endogenous NTR can catalyze the enzymatic cleavage reaction
of compound 2 to compound 3 in living tumor cells, and the
formed compound 3 can localize in the lysosome. Based on
these results, we speculate that the cell apoptosis was because
the intracellular lysosomes were destroyed by the 1O2
generated in situ. To prove the intracellular lysosomes lysis,
acridine orange (AO) was used as the indicator for lysosomal
integrity.57,59 As shown in Figure 6, the red fluorescence which
represents the integrity of lysosomes disappeared only in the
group of the cells irradiated by 630 nm LED under 10%
oxygen concentration. This result indicates that the lysosomes
To make sure that the cell death was caused by the
generation of 1O2, we use 2,7-dichlorodihydrofluorescein
1
diacetate (DCFH-HA) as a O2 probe to further prove the
51
1
intracellular O2 generation by confocal imaging. As shown
1
in Figure S18, the green fluorescence suggests that O2 was
generated under hypoxia conditions with irradiation. At the
same time, it is obvious that 10% oxygen concentration gives
1
the best O2 generation yield according to the bright green
fluorescence intensity.
In addition, the activated PDT efficiency by mild hypoxia of
10% oxygen concentration was confirmed using a confocal
fluorescence image and the fluorescence-activated cell sorting
(FACS) statistical analysis. By comparing images c1 with c3 for
non-irradiation cells in Figure 4c, we found that the mild
hypoxic cells have a similar good survival state with the
normoxic cells without photoirradiation, considering that
calcein AM is a living-cell staining dye with green
fluorescence.26,51−54 By comparing images c1 with c2 for
normoxic cells, we found that the photoirradiation did not hurt
the cells. Since no green fluorescence was observed in image c4
for the mild hypoxic cells with photoirradiation, these results
indicate that the PDT efficiency can be activated only when
the two demands of photoirradiation and mild hypoxia are
met. According to the FACS statistical analysis shown in
Figure 4d, the percentage of apoptosis (46.6%) under mild
hypoxia conditions was much higher than that (0.05%) under
normoxia conditions for the HeLa cells with photoirradiation.
FACS statistical analysis not only confirmed that the PDT
efficiency of compound 2 can be activated by mild hypoxia but
also suggested that the PDT-induced cell death is via
apoptosis, not necrosis.55,56
1
were destroyed by the O2 generated in situ.
Fluorescence and PDT Activation of Compound 2 in
Tumor-Bearing Mice. After the NTR-activated PDT
performance of compound 2 was verified in the living cells,
we try to explore how the activation can be fulfilled in vivo.
Anesthetized SCID/BALB/c-nude mice were chosen as the
mammal model. The fluorescence response of compound 2
toward endogenous NTR was tested in HeLa tumor-bearing
mice. After the tumor model formed with a 1.0 cm diameter,
one group was injected with compound 2 and the other group
was injected with compound 2 and dicoumarin. The time-
dependent fluorescence images and intensity were collected
every 5 min after injection. As shown in Figure 7, there was an
obvious distinction in the fluorescence brightness between
these two groups due to the inhibition effect of dicoumarin.
These results indicate that endogenous NTR in the tumor area
can efficiently switch on the fluorescence of compound 2. The
fluorescence intensity reached a maximum around 30 min after
injection and leveled off until 80 min, which is still a quick
response time considering the hysteresis effect due to the
diffusion process of the sensing probe. From the confocal
images of the tumor slices dissected at 80 min (Figure S19),
the distinct red fluorescence is observed in the whole image.
This result indicates that compound 2 was distributed evenly
inside the whole tumor after 80 min, which gives PDT a good
working time zone. Meanwhile, since the extent of hypoxia
should become mild at the edge of the solid tumor, this result
To further investigate the therapeutic mechanism of
compound 2-mediated PDT, a subcellular colocalization
experiment of compound 2 was performed by co-staining
with the organelle-specific fluorescent dye lysosome green, a
commercial lysosome tracker.51,57,58 As shown in Figure 5, the
Pearson’s correlation coefficient is up to 0.93, which means the
compound with red fluorescence is localized in the lysosome.
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with hematoxylin and eosin (H&E) after the mice were
sacrificed. As shown in Figure 8d, significant apoptosis can
only be observed in the positive tumor sections. All these
results support that compound 2 has high activatable
phototoxicity and a low side effect, which makes it possible
to differentiate the nidus region from the surrounding healthy
tissues and site-specifically inactivate tumor cells.
CONCLUSIONS
■
In summary, we have synthesized two smart activatable
theranostic molecules 1 and 2 based on compound 3, a
TADF fluorescein derivative. Compound 2 has better
selectivity and response rate than compound 1 when sensing
NTR. Importantly, the PDT efficiency of the selected
compound 2 can be efficiently activated by the mild hypoxia
microenvironment of 10% oxygen concentration. During the
activation, compound 3 can be generated in situ from
compound 2. The formed compound 3 has a long triplet
state lifetime (16.3 μs), which leads to efficient production of
1O2. The NTR-activation process was verified to be performed
under mild hypoxia in living HeLa cells and in vivo animals. To
our delight, it was found that the PDT efficiency of compound
2 under hypoxia conditions (10% oxygen concentration) was
much better than under normoxia (21% oxygen concen-
tration). In fact, compound 2 under mild hypoxia has a high
PDT efficiency, even better than PpIX, a traditional photo-
sensitizer. The excellent PDT effect was associated with its
lysosomal targeting ability. Besides the NTR-activatable
theranostic properties, compound 2 also possesses many
other advantages such as two-photon excitation, large Stokes
shift, short response time, and high specificity and sensitivity.
Importantly, the strategy of targeting the mild hypoxic cells in
the edge of tumor tissue could heal the Achilles’ heel of
traditional PDT. We believe that this smart theranostic
molecule has high potential to be applied in clinical
investigation as a theranostic anticancer prodrug.
Figure 8. PDT efficiency in HeLa tumor mice with 590 nm LED (16
mW/cm2). (a) Photos of the four groups of tumor-bearing mice. (b)
Changes of tumor volumes of the four groups. Error bars indicate SD
(n = 4), the date was determined using Sidak’s multiple comparison
tests. (c) Changes of the weight of the mice through the phototoxicity
test. (d) H&E staining of tumor tissue sections (from left to right, (1)
saline, (2) Irradiation, (3) compound 2 (0.1 mM, 100 μL) without
irradiation, (4) compound 2 (0.1 mM, 100 μL) with irradiation). The
scale is 50 μm.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.9b04488.
■
S
Synthesis of compounds 1−3, supporting data for the
activation mechanism, docking calculations of com-
pounds 1−2 with NTR, fluorescence spectra, lumines-
cence lifetime, confocal fluorescence imaging (PDF)
also confirms that NTR can be induced even under mild
hypoxia to trigger the transfer from compound 2 to 3. It means
that in vivo PDT will have a good oxygen concentration
around 10% to achieve high efficiency.
AUTHOR INFORMATION
Corresponding Author
*E-mail: songfl@dlut.edu.cn, songfl@sdu.edu.cn.
■
Finally, we checked the high PDT performance of
compound 2 in HeLa tumor-bearing mice. The mice were
randomly divided into four groups. As shown in Figure 8a,b,
the tumor growth of mice under the combination conditions of
existing compound 2 and irradiation was completely inhibited,
whereas all the control mice of the other three groups
exhibited no obvious therapeutic effect. Besides, no significant
changes could be found from the weights of all these four
groups of mice (Figure 8c) and there was no obvious change in
the pericancerous skin tissue before and after treatment as
shown in Figure S20. It means that the side effects of both
compound 2 and irradiation were negligible. To further verify
the PDT efficiency in mice, the tumor sections were stained
ORCID
Fengling Song: 0000-0001-5305-676X
Wenbo Shi: 0000-0003-2033-334X
Bo Song: 0000-0002-8584-1591
Xiaojun Peng: 0000-0002-8806-322X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by the National Natural
Science Foundation of China (21877011, 21576038,
21421005), the Fundamental Research Funds for the Central
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