Developing glutathione-activated catechol-type diphenylpolyenes as small molecule-based and mitochondria-targeted prooxidative anticancer theranostic prodrugs

Article abstract of DOI:10.1016/j.freeradbiomed.2019.01.033

Developing concise theranostic prodrugs is highly desirable for personalized and precision cancer therapy. Herein we used the glutathione (GSH)-mediated conversion of 2,4-dinitrobenzenesulfonates to phenols to protect a catechol moiety and developed stable pro-catechol-type diphenylpolyenes as small molecule-based prooxidative anticancer theranostic prodrugs. These molecules were synthesized via a modular route allowing creation of various pro-catechol-type diphenylpolyenes. As a typical representative, PDHH demonstrated three unique advantages: (1) capable of exploiting increased levels of GSH in cancer cells to in situ release a catechol moiety followed by its in situ oxidation to o-quinone, leading to preferential redox imbalance (including generation of H₂O₂ and depletion of GSH) and final selective killing of cancer cells over normal cells, and is also superior to 5-fluorouracil and doxorubicin, the widely used chemotherapy drugs, in terms of its ability to kill preferentially human colon cancer SW620 cells (IC₅₀ = 4.3 μM) over human normal liver L02 cells (IC₅₀ = 42.3 μM) with a favourable in vitro selectivity index of 9.8; (2) permitting a turn-on fluorescent monitoring for its release, targeting mitochondria and therapeutic efficacy without the need of introducing additional fluorophores after its activation by GSH in cancer cells; (3) efficiently targeting mitochondria without the need of introducing additional mitochondria-directed groups.

Full text of DOI:10.1016/j.freeradbiomed.2019.01.033

Accepted Manuscript
Developing glutathione-activated catechol-type diphenylpolyenes as small molecule-
based and mitochondria-targeted prooxidative anticancer theranostic prodrugs
Xia-Zhen Bao, Fang Dai, Qi Wang, Xiao-Ling Jin, Bo Zhou
PII:
S0891-5849(18)31521-1
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.01.033
FRB 14131
Reference:
To appear in: Free Radical Biology and Medicine
Received Date: 1 September 2018
Revised Date: 24 January 2019
Accepted Date: 24 January 2019
Please cite this article as: X.-Z. Bao, F. Dai, Q. Wang, X.-L. Jin, B. Zhou, Developing glutathione-
activated catechol-type diphenylpolyenes as small molecule-based and mitochondria-targeted
prooxidative anticancer theranostic prodrugs, Free Radical Biology and Medicine (2019), doi: https://
doi.org/10.1016/j.freeradbiomed.2019.01.033.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
Developing glutathione-activated catechol-type diphenylpolyenes as
small molecule-based and mitochondria-targeted prooxidative
anticancer theranostic prodrugs
*
Xia-Zhen Bao, Fang Dai, Qi Wang, Xiao-Ling Jin and Bo Zhou
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222
Tianshui Street S., Lanzhou, Gansu 730000, China
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
1
1
1
1
1
1
1
1
1
1
2
2
2
__________________
*Corresponding author.
E-mail: bozhou@lzu.edu.cn
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
ABSTRACT
Developing concise theranostic prodrugs is highly desirable for personalized and
precision cancer therapy. Herein we used the glutathione (GSH)-mediated conversion
of 2,4-dinitrobenzenesulfonates to phenols to protect a catechol moiety and developed
stable pro-catechol-type diphenylpolyenes as small molecule-based prooxidative
anticancer theranostic prodrugs. These molecules were synthesized via a modular
route allowing creation of various pro-catechol-type diphenylpolyenes. As a typical
representative, PDHH demonstrated three unique advantages: (1) capable of
exploiting increased levels of GSH in cancer cells to in situ release a catechol moiety
followed by its in situ oxidation to o-quinone, leading to preferential redox imbalance
(including generation of H O and depletion of GSH) and final selective killing of
2
2
cancer cells over normal cells, and is also superior to 5-fluorouracil and doxorubicin,
the widely used chemotherapy drugs, in terms of its ability to kill preferentially
human colon cancer SW620 cells (IC = 4.3 µM) over human normal liver L02 cells
5
0
(IC = 42.3 µM) with a favourable in vitro selectivity index of 9.8; (2) permitting a
5
0
turn-on fluorescent monitoring for its release, targeting mitochondria and therapeutic
efficacy without the need of introducing additional fluorophores after its activation by
GSH in cancer cells; (3) efficiently targeting mitochondria without the need of
introducing additional mitochondria-directed groups.
Keywords: Glutathione; Diphenylpolyenes; Prooxidant; Theranostic prodrug;
Mitochondria-targeted
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1. Introduction
The development of theranostic agents is particularly attractive in the context of
personalized and precision cancer therapy on account of their dual ability to offer both
diagnosis and therapy [1,2]. Prodrugs have also attracted much attention due to their
conversion to toxic species only at the cancer-specific conditions (such as low pH [3],
increased levels of reactive oxygen species (ROS) [4-7], reduced glutathione (GSH)
[8-12] and overexpressed enzymes [13,14]), thereby potentially lacking the dangerous
side effect. With the use of the two concepts, several types of theranostic prodrugs
equipped with fluorophores as optical reporters have been successfully explored to
allow facile monitoring of the release, location and efficacy of anticancer agents [2].
However, they are almost bulky combined systems consist of masked anticancer
drugs, cleavable linkers, fluorophores and cancer targeting ligand. Especially,
additional conjugation of a fluorescent tag is required because most of the anticancer
drugs are intrinsically nonfluorescent. Such modifications might not make sure the
fluorescence is correlated with real location of the drugs once they are released [15].
Therefore, developing small molecule-based anticancer theranostic prodrugs without
the need of introducing additional fluorophores remains a challenge and is highly
desirable to track them and directly visualize their therapeutic effects.
Compared with normal cells, cancer cells exhibit aberrant redox homeostasis: on
the one hand, they need increased levels of ROS to maintain their malignant
phenotypes; on the other hand, they survive the ROS stress by upregulating GSH
synthesis [16-19]. The aberrant redox homeostasis illustrates redox Achilles heel of
3

ACCEPTED MANUSCRIPT
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
cancer cells [20,21], because as a kind of already stressed cells they are more
vulnerable to further generation of ROS than normal cells [16-19]. Thus, developing
prooxidative anticancer agents (PAAs) capable of generating ROS or/and depleting
GSH has attracted increasing attention [16-19]. Indeed PAAs can preferentially kill
cancer cells over normal cells in this way, but they also increase the ROS amount in
normal cells, leading to side effects [22]. An efficient means to solve the flaw is
designing prooxidative anticancer prodrugs that can be selectively activated by redox
Achilles heel of cancer cells to induce highly selective generation of ROS in cancer
cells, as depicted in Scheme 1A.
Catechol moiety, found widely in nature, appears to be an important component to
construct dietary natural products such as piceatannol, green tea polyphenols,
quercetin, caffeic acid, delphinidin, hydroxytyrosol and so on. These catechol-type
molecules usually hold promise in cancer chemoprevention at least in part due to their
prooxidative role based on their oxidative conversion into o-quinones during which
ROS are generated [23-29]. However, their oxidative instability also constitutes the
main drawback to use them as anticancer agents.
Stilbenes, as the simplest diphenylpolyenes, are a small family of plant secondary
metabolites as exemplified by the most extensively studied resveratrol [30]
pterostilbene and piceatannol. We found previously that among resveratrol and its
hydroxylated analogs, a catechol-type stilbene (3,4-dihydroxy-trans-stilbene, DHS) is
the only one that can exploit intracellular copper ions to in situ generate its
corresponding o-quinone, resulting in activation of nuclear factor erythroid-2 related
4

ACCEPTED MANUSCRIPT
8
9
9
9
9
9
9
9
9
9
9
9
0
1
2
3
4
5
6
7
8
9
factor 2 (Nrf2) [26], an important target for cancer chemoprevention. We believe that
further elongating the conjugated links of DHS could facilitate its oxidation to
o-quinone and development of the catechol-based PAAs, for example, inserting of
additional double bonds between two aromatic rings to generate various
diphenylpolyenes (Scheme 1B). Nevertheless, this modification will intensify their
instability, a bottleneck in practical use of catechol molecules as mentioned above.
Scheme 1 here
In our opinion, the bottleneck could be solved by designing pro-catechols that is
usually robust but can be in situ activated to catechols under cancer-specific
conditions, such as increased levels of GSH observed in cancer cells [31,32]. It is
well-established that 2,4-dinitrobenzenesulfonates (DNBS) are easily subject to
nucleophilic attack by thiols including GSH followed by exposure of a free phenolic
hydroxyl group [33]. This reaction prompts the development in designing fluorescent
probes for thiols [34,35], GSH-activated photosensitizers for selective photodynamic
action [36,37] and thiols-triggered anticancer prodrug [38]. However, this reaction, to
date, has not been used for protecting a catechol moiety.
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
As part of our effort to develop natural product-inspired PAAs for targeting redox
Achilles heel of cancer cells [26-29, 39-43], herein we used the above GSH-mediated
conversion of DNBS to phenols as a general strategy to design a variety of
pro-catechol-type diphenylpolyenes (PDHS, PDHB and PDHH differentiated by
various numbers of double bonds between two aromatic rings, Scheme 1B and
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Scheme 2) as new-style theranosic prodrugs. Specifically, the 4-OH on their catechol
moieties was selectively blocked by DNBS since this hydroxyl is more reactive than
3-OH. We reasoned that the masked 4-OH could be activated by GSH in cancer cells
to in situ release a catechol moiety, while maintaining its salient feature in normal
cells; the resulting catechol moiety allows its in situ oxidation to o-quinone, leading to
generation of ROS and final selective killing of cancer cells (Scheme 1B). The
designed diphenylpolyenes also serve as the reporters of their release, location and
therapeutic efficacy because their fluorescence is mostly quenched by attachment of a
strong electron withdrawing DNBS, but once they are activated by GSH in cancer
cells, permitting a turn-on fluorescent monitoring. Moreover, the DNBS works as a
cleavable linker under a cancer-specific condition (increased levels of GSH), the total
theranostic prodrug system is thereby very concise. Unexpectedly, the
organelle-specific delivery was found in the case of diphenylpolyenes which were
efficiently localized within mitochondria, providing novel mitochondria-directed
groups that would facilitate developing mitochondria-targeted fluorescence probes
and drugs.
2. Materials and methods
2.1. Materials
Leibovitz’s L-15 Medium (L-15), Roswell Park Memorial Institute (RPMI)-1640,
Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Media (MEM),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum
6

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
albumin (BSA), dihydroethidium (DHE), propidium iodide (PI), rhodamine 123, yeast
glutathione reductase (GR), 2-vinylpyridine (97%), reduced L-glutathione (GSH),
oxidized L-glutathione (GSSG), L-buthionine-(S,R)-sulfoximine (BSO), catalase
from
5’-dithiobis-2-nitrobenzoicacid (DTNB), glutathione S-transferase (GST) from equine
liver, ethacrynic acid (EA) and carbonyl cyanide
bovine
liver
(CAT),
DL-dithiothreitol
(DTT)
and
5,
4-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Mito-Tracker Deep Red and Amplex™ Red Hydrogen
Peroxide/Peroxidase Assay Kit were purchased from Thermo Fisher Scientific
(Waltham, USA). Annexin V-FITC Apoptosis Detection Kit was from BD (Becton,
Dickinson and Company). Nicotinamide adenine dinucleotide 2’-phosphate reduced
tetrasodium salt (NADPH) was from Roche Diagnostics GmbH (Mannheim,
Germany). ER-Tracker Red, Lyso-Tracker Red and bicinchoninic acid (BCA) protein
assay kit were from Beyotime Institute of Biotechnology (Jiangsu, China).
2.2. Synthesis of the catechols and the pro-catechols
The synthesis details of the catechols and the pro-catechols were shown in the
1
13
Supplemental Information. These compounds were characterized with H NMR, C
NMR and HRMS spectra, and their purity was further checked by HPLC-based
analyses.
2.3. Stability assay
7

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Stability of the catechols and pro-catechols in PBS/DMSO (1/1, v/v, pH 7.4) at
room temperature was monitored at their maximum absorbance for 10 h (at 30 min
intervals) by using a UV/visible spectrophotometer. Additionally, their stability was
further validated using HPLC-based analyses. Specifically, the solutions were
prepared by dissolving the catechols or the pro-catechols in PBS/DMSO (1/1, v/v) to
a final concentration of 160 ꢀM, then were maintained at room temperature for
different periods of time for HPLC analysis using a Waters HPLC system equipped
with a Sun Fire™, C-18 column (4.6 mm × 150 mm). H O (eluent A)/CH CN (eluent
2
3
B) was used as the mobile phase. The temperature of the column was maintained at 35
°C and the flow rate of the mobile phase was 1.0 mL/min. The run was set as follows:
for PDHS A(H O): B(CH CN) = 0.45:0.55, for PDHB A(H O): B(CH CN) = 0.4:0.6,
2
3
2
3
for PDHH A(H O): B(CH CN) = 0.35:0.65.
2
3
2.4. HPLC-based analyses for in vitro activation of the pro-catechols by GSH
The ability of the pro-catechols to release its parent catechols in the presence of
GSH at ambient temperature was evaluated using a Waters HPLC system equipped
with a Sun Fire™, C-18 column (4.6 mm × 150 mm). After adding GSH (1.6 mM) to
a solution of pro-catechols (160 ꢀM) in CH CN/H O (1/1, v/v), the products at
3
2
different periods of time (1, 3, 6, 9 and 12 h) were examined by HPLC. The
experimental conditions were the same as those described in the stability assay using
HPLC.
8

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
2.5. Fluorometric analysis for in vitro activation of the pro-catechols by GSH
Fluorescence spectroscopic studies were carried out at room temperature on a LS
55 Luminescence Spectrometer (PerkinElmer). The catechols and the pro-catechols
were dissolved in DMSO to make a 3 mM stock solution, which was diluted to 1 ꢀM
as testing solution in PBS/DMSO (8/2, v/v) for measurement.
2.6. Kinetic analysis for in vitro activation of the pro-catechols by GSH
The reactions of the pro-catechols (5 ꢀM) with GSH (0.25, 0.5 and 1 mM) were
carried out in PBS/DMSO (v/v, 1/1) under pseudo-first-order conditions at 37 °C in
the absence or presence of equine liver GST (0.03 mg/mL). The increased
fluorescence due to the production of the catechols, was monitored within 1 h using
TECAN Infinite M200 multimode microplate reader (For DHS, λex = 330 nm, λem =
410 nm; For DHB, λex = 350 nm, λem = 440 nm; For DHH, λex = 370 nm, λem =
475 nm). The second-order rate constants (k ) were obtained by plotting the
2
pseudo-first-order constants versus the corresponding GSH concentration.
2.7. Cell culture
Human colon cancer SW620, malignant melanoma A375, ovarian cancer SKOV3,
and fibrosarcoma HT-1080 cells as well as normal cells including human liver L02,
human umbilical vein endothelial HUVEC and mouse hippocampal neuronal HT-22
cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences. SW620 cells were incubated in L15 medium
9

ACCEPTED MANUSCRIPT
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
supplemented with 10% Newborn Calf Serum (NCS) and gentamycin sulfate (50
ꢀg/L); A375 and HT-22 cells were cultured in DMEM supplemented with 10% NCS,
NaHCO (3.7 g/L), L-Glutamine (0.2 g/L) and antibiotics (penicillin/streptomycin,
3
100 kU/L); SKOV3, L02 and HUVEC cells were cultured in RPMI-1640
supplemented with 10% fetal bovine serum (FBS), NaHCO (2 g/L) and antibiotics
3
(penicillin/streptomycin, 100 kU/L); HT-1080 cells was grown in MEM supplemented
with 10% (v/v) FBS, penicillin (100 kU/L), streptomycin (100 kU/L), NaHCO (2.2
3
g/L) and sodium pyruvate (0.11 g/L). The cells were maintained in a humidified
incubator in 5% CO /95% air at 37 °C and the exponentially growing cells were used
2
for all experiments.
2.8. MTT assays
4
Cells were plated in flat-bottomed 96-well plates (2×10 cells/well for SW620,
3
4×10 cells/well for A375, SKOV3, HT-1080, L02, HUVEC and HT-22) and
incubated at 37 °C for 24 h. The cells were treated with the tested compounds at
varying concentrations and incubated for 48 h at 37 °C. In the case of inhibition, the
cells were pretreated with DTT (400 ꢀM), CAT (0.5 mg/mL) or EA (10 ꢀM) for 1 h
before adding the tested compounds. After 48 h exposure, medium was replaced by
100 µL of MTT solution (0.5 mg/mL in PBS) and incubation for another 4 h. The
MTT medium was aspirated and substituted with 100 µL DMSO. UV-vis absorbance
was measured at 570 nm using a microplate reader (Bio-Rad M680 ELISA).
Experiments were performed each in sextuplicate and repeated three times.
1
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
2.9. Fluorescence imaging for subcellular distribution and therapeutic efficacy
5
SW620 and L02 cells were seeded into 6-well plates at 4 × 10 cells/well and
cultured at 37 °C with 5% CO for 24 h. The cells were incubated with PDHH (5 ꢀM)
2
for 1, 2 and 3 h. In the case of inhibition, SW620 cells were pretreated with BSO (1
mM) for 1 h before incubation with PDHH. For determining the subcellular
distribution of the catechols and the pro-catechols, SW620 cells were incubated with
the tested compounds for 3 h. Then the cells were washed with PBS followed by
incubation with Mito-Tracker Deep Red (1.0 µM), ER-Tracker Red (1 ꢀM) or
Lyso-Tracker Red (1 ꢀM) for another 30 min. For monitoring the death of SW620
cells induced by PDHH, the cells were treated with PDHH (15 ꢀM) for 9, 12, 18 and
24 h, then the cells were collected and labeled with PI (5 ꢀg/mL) for 30 min. All the
cells were washed by PBS buffer before imaging. The fluorescence images were
acquired by microscope Leica DM 400B (Leica Microsystems CMS GmbH, Wetzlar,
Germany). All experimental parameters (exposure time, objective lens) remained
unchanged when the different fluorescence images were captured.
2.10. Fluorescence imaging for generation of intracellular ROS
5
SW620 cells and L02 cells were seeded onto 6-well plates at 4×10 cells/well. After
24 h, the cells were incubated with 5 ꢀM PDHH for 6 h or 9 h. Then the cells were
rinsed with PBS for 2-3 times and stained with 3 ꢀM DHE for additional 30 min at
37°C. Then the cells were washed by PBS buffer before imaging. The fluorescence
images were acquired by microscope Leica DM 4000B (Leica Microsystems CMS
1
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
GmbH, Wetzlar, Germany) with a×40 objective lens.
2.11. Measuring H O released from SW620 cells
2
2
4
SW620 (2×10 cells/well) cells were seeded in 96-well plates overnight and the
medium was replaced by Krebs-Ringer phosphate (KRPG) buffer before the cells
were treated with PDHH (2 and 5 ꢀM) for 6 and 9 h in the absence or presence of 1 h
pretreatment with CAT. Aliquots of medium were taken out and their H O levels
2
2
were determined using the Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit
[44]. Specifically, 20 ꢀL of medium was added into 100 ꢀL of reaction mixture
(containing 50 µM Amplex Red reagent and 0.1 U/mL HRP in KRPG buffer),
warmed at 37 °C for ten minutes, and then subjected to fluorescence analysis on a
TECAN Infinite M200 multimode microplate reader (λex = 530 nm, λem = 590 nm)
with 20 ꢀL of KRPG buffer as the negative control [44].
2.12. Detection of intracellular GSH levels
5
5
SW620 (4×10 cells/well) and L02 (4×10 cells/well) cells were seeded in 6-well
plates in growth medium (2 mL) and then incubated at 37°C in a 5% carbon dioxide
atmosphere. Intracellular GSH contents were evaluated using the glutathione
reductase-DTNB recycling assay [45], and the related details were described in our
previous paper [43].
2.13. Measurement of mitochondrial membrane potential
1
2

ACCEPTED MANUSCRIPT
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
SW620 and L02 cells were seeded onto 6-well plates at a density of 4×10 cells per
well and then incubated with FCCP or PDHH for the 18 and 24 h. The cells without
treatment were used as the control. After incubation, the cells were trypsinized and
washed with PBS followed by staining with 5 ꢀM of rhodamine-123 for additional 30
min at 37°C. After washing again with PBS, the cells were analyzed using
FACSCanto flow cytometer (Becton Dickinson, San Jose, CA, USA). The mean
fluorescence was determined by counting 10,000 events.
2.14. Apoptosis analysis
5
5
SW620 (4×10 cells/well) and L02 (4×10 cells/well) cells were seeded on each
well of a six-well plate and allowed to grow overnight. Medium was changed and the
cells were treated with serial concentrations of PDHH for 48 h at 37 °C. Then the
cells were trypsinized, washed repeatedly with PBS for three times and centrifuged at
350 g for 5 min. The cells were resuspended in 100 µL 1×Annexin V binding buffer,
labeled with 5 µL annexin V-FITC/PI from the detection kit (Becton Dickinson, San
Jose, CA, USA), and incubated for 15 min at room temperature. After incubation, 400
µL of 1×Annexin V binding buffer was added to each sample prior to analyses on the
FACSCanto flow cytometer (Becton Dickinson, San Jose, CA, USA). A total of
4
1.0×10 events were counted for each sample.
2.15. Statistical analysis
Data are expressed as the mean ± SD. Statistical comparisons among the results
1
3

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
were performed using analysis of variance. Significant differences (P < 0.05) between
the means of two groups were analyzed by Student's t-test.
3. Results and discussion
3.1. Synthesis of the diphenylpolyenes
The designed diphenylpolyenes were synthesized according to the optimized routes
as outlined in Scheme 2. The requisite aldehyde 2 was obtained by a sequential
protection of the 4- and 3-OH of 3,4-dihydroxybenzaldehyde using
t-butyldimethylsilyl (TBS) and methoxymethyl (MOM) ethers, respectively. Another
key intermediate benzylphosphonate 6 was prepared from the conjugate carboxylic
acid via esterification, reduction, bromination and nucleophilic substitution. With the
intermediates 2 and 6 in hand, the key Witting-Hornor reaction was explored under
various alkaline conditions (including NaH/THF, MeONa/DMF, LDA/THF and
t-BuOK/DMF). The reaction proceeded smoothly only in t-BuOK/DMF, giving
surprisingly the product 7 with deprotection of TBS in moderate yield. To the best of
our knowledge, this is the first use of t-BuOK to complete one-step
Witting-Hornor/selective-deprotection of TBS reaction. Treatment of 7 with
2,4-dinitrobenzenesulfonyl chloride in DCM and Et N followed by deprotection of
3
MOM under the con. HCl condition afforded finally the designed diphenylpolyenes 9
(PDHS, PDHB and PDHH) in overall yields of 30.5, 16.5 and 14.6 %, respectively.
Noticeably, the total synthesis was accomplished via a modular route allowing
1
4

ACCEPTED MANUSCRIPT
creation of various pro-catechol-type diphenylpolyenes. In addition, the
2,4-dinitrobenzenesulfonyl group was added at the final stage of the synthesis, so that
this synthesis procedure may be applied to create other protecting group onto the
catecholic 4-OH.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Schemes 2-3 here
3.2. Stability analysis
The stability of the catechols and pro-catechols was first evaluated by monitoring
their UV/Vis absorption changes in PBS/DMSO (1:1, v/v) at ambient temperature (see
Supplementary Figure S1). Figure 1A and B shows the changes in absorbance at
maximum as a function of the incubation time. The catechols (DHS, DHB and DHH)
were less stable due to their oxidative susceptibility, as suggested by their gradual
decay over time, and their decay rates were increased as elongation of the conjugated
links. In contrast, the pro-catechols (PDHS, PDHB and PDHH) showed almost no
signs of decay in PBS for 10 h, highlighting their improving oxidative stability by
blocking the 4-OH. The stability was also further validated by HPLC-based analyses
(Figure 1C and D). Also, the pro-catechols demonstrated significantly better stability
than the catechols. Noticeably, the pro-catechols showed a handful of decay probably
owing to their hydrolysis to the catechols. The decay was not observed based on
UV-visible spectral analyses because the pro-catechols and the catechols exhibited
almost identical absorbance characteristics.
Figure 1 here
1
5

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
3.3. In vitro activation of pro-catechols by GSH
The in vitro activation of the pro-catechols by GSH was next monitored using
HPLC-based analyses. Addition of 10 equiv. of GSH to a solution of PDHH in
CH CN/water (1/1, v/v) induced a decrease in its peak intensity at 14.69 min
3
accompanied by the appearance of a new chromatographic peak at 4.77 min
corresponding to the formation of DHH (Figure 2). Similar activation was also
observed in the case of PDHS and PDHB (Figure 2). It should be pointed out that the
build-up of the catechols is not completely correlated with the decay of the
procatechins due to oxidative instability of the former (Figure 2B, D and F).
Figure 2 here
The in vitro activation of the pro-catechols was also checked by fluorescence
analyses. As expected, the pro-catechols compared with the parent catechols exhibited
a very weak fluorescence emission in PBS/DMSO (8:2, v/v) but the signals were
significantly increased over time upon adding GSH, suggesting a turn-on fluorescence
response (Figure 3). Furthermore, the fluorescence emission spectra generated by the
reaction of pro-catechols with GSH were consistent with those of the corresponding
catechols (Figure S2), supporting strongly activation of the pro-catechols to the
corresponding catechols by GSH. It is also noticeable that the degradation of the most
o
unstable DHH within 1 h in PBS/DMSO (v/v, 1:1) at 37 C was almost completely
inhibited by a large excess of GSH (Figure S3). This facilitates the investigation on
1
6

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
the reaction kinetics of the pro-catechols with GSH based on the increased
fluorescence emission of the resulting catechols. Consequently, we used the
pseudo-first-order conditions to determine the second-order rate constants (k ) of the
2
pro-catechols with GSH, by using a multimode microplate reader. Plotting the
pseudo-first-order rate constants (k ) against the GSH concentrations gave the
obs
straight lines with an intercept above the zero point, indicating a possible more
complex kinetics (Figure 4). On the basis of the k values (Table 1) obtained from the
2
straight lines, the reactivity of the pro-catechols with GSH follows the sequence of
PDHH > PDHB > PDHS, highlighting that the reactivity was increased as elongation
of the conjugated links. Considering that glutathione S-transferases (GSTs) as phase II
detoxification enzymes catalyze the nucleophilic attack by GSH on non-polar
electrophilic molecules [46], we also compared the k values in the absence and
2
presence of equine liver GST to clarify its contribution on the activation of
pro-catechols by GSH. It can be seen from Table 1 that the presence of GST enhanced
moderately the k values of PDHB and PDHS, and failed to do this in the case of
2
PDHH, suggesting that they are not efficient substrates of GST. Taken together, the
above HPLC and fluorescence data support strongly the activation of the
pro-catechols by GSH to release the corresponding catechols.
Figures 3 and 4 and Table 1 here
3.4. Cytotoxicity and structure-activity relationship
Subsequently, MTT assay was performed to assess the cytotoxicity of all tested
1
7

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
molecules towards various cancer cells (human colon cancer SW620, malignant
melanoma A375, ovarian cancer SKOV3 and fibrosarcoma HT-1080 cells) and
normal cells (human liver L02, human umbilical vein endothelial HUVEC and mouse
hippocampal neuronal HT-22 cells). The IC values derived from the titration curves
5
0
(Figure S4) are listed in Table 2 and allowed us to identify the following
structure-activity relationships: (1) the cytotoxicity was obviously increased as
elongation of the conjugated links in both the catechols and pro-catechols, in line with
their ability to facilitate the formation of o-quinone; (2) the pro-catechols exhibited
higher cytotoxicity and better selectivity than the corresponding catechols with PDHH
being the most active one among the tested molecules; For example, in SW620 and
L-02 cells, IC50 values of PDHH were determined as 4.3 and 42.3 µM (selectivity
index: 9.8), whereas those of DHH were 9.4 and 33.2 µM, respectively; (3) noticeably,
PDHH was superior to 5-fluorouracil and doxorubicin, the widely used chemotherapy
drugs, in the ability to kill preferentially SW620 over normal L-02 cells with a
favourable in vitro selectivity index of 9.8.
Table 2 here
3.5. Visualization for the selective activation of PDHH by GSH in SW620 cells over
normal L02 cells
The increased selective cytotoxicity of the pro-catechols compared with the
corresponding catechols are in tune with their improving stability and hint at the
possibility that they could be selctively activated by GSH in cancer cells to in situ
1
8

ACCEPTED MANUSCRIPT
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
release the catechol moiety. To further clarify this point, we selected the most active
PDHH to observe its selective activation in live SW620 cells over normal L02 cells
by fluorescence imaging. After incubation of SW620 cells with PDHH, we observed a
gradual increased bright-blue fluorescence signal over time (Figure 5A-D). However,
this signal was significantly abrogated by pretreatment of the cells with
L-buthionine-sulfoximine (BSO) for 1 h (Figure 5E), a GSH synthase inhibitor,
confirming that activation of PDHH is GSH-dependent. In comparison, only faint
activation of PDHH occurred in L02 cells after 6 h incubation (Figure 5F-I). The
significant difference in activating PDHH between SW620 and L02 cells is a result of
their different basal levels of GSH [31,32], and also the reason why PDHH could kill
preferentially SW620 over L-02 cells. This is further supported from our determined
basal levels of GSH of SW620 and L02 cells (122 ± 1.92 and 29 ± 3.18 nmol/mg
protein, respectively) by the glutathione reductase-DTNB recycling assay [45]. In
other words, the 4.2-fold difference in the basal levels of GSH between the two cell
lines might be enough for making the selective cytotoxicity of PDHH (selectivity
index: 9.8). More importantly, in line with the abrogation of the fluorescence signal,
pretreatment of SW620 cells with BSO for 1 h decreased siginificantly the basal
levels of GSH from the original 122 ± 1.92 to 55 ± 2.92 nmol/mg protein.
Figure 5 here
GST is often overexpressed in numerous cancer cells and contributes to resistance
of cancer cells towards cytostatic drugs by catalyzing their conjugation with GSH [46].
1
9

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
To clarify whether GST is involved in the activation of PDHH by GSH and
contributes to its selective cytotoxicity, we further checked the effect of ethacrynic
acid (EA, a GST inhibitor) on the cytotoxicity of PDHH against SW620 cells. As
illustrated in Figure S5, pretreatment of SW620 cells with EA had no appreciable
effect on the cytotoxicity of PDHH. This is consistent with the kinetic results showing
that GST has no impact on the reaction of PDHH with GSH (Table 1), and further
supports that the selective cytotoxicity of PDHH is caused by the difference of GSH
levels, rather than that of GST levels, between SW620 and L02 cells. Furthermore,
previous research has revealed that uptake of resveratrol, a hydroxylated stilbene, by
human hepatocytes is similar to that obtained in human hepatoma HepG2 cells [47].
So that we speculated that the selectivity observed in the case of PDHH as the same
diphenylpolyene with resveratrol is not due to the differential cellular uptake.
According to the above results, we can conclude that PDHH can be selectively
activated by increased levels of GSH in SW620 cells to in situ release a catechol
moiety, resulting in its selective cytotoxicity, and its activation can be visualized by a
“turn-on” blue fluorescence response. In addition, it should be emphasized that the
byproducts generated in the activation of PDHH by GSH, SO and (2,4-dinitrophenyl)
2
glutathione, were non-cytotoxic, as described previously [39].
3.6. Visualization for the subcellular distribution of PDHH after activation
To determine the subcellular distribution of PDHH after activation, co-localization
experiments were performed. SW620 cells were co-stained with PDHH and
2
0

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
commercially available Mito-Tracker Deep Red, ER-Tracker Red or Lyso-Tracker
Red. As shown in Figure 6, PDHH was gradually activated in SW620 cells over the
course of 3 h and the blue fluorescence signal at the time points of 3 h was
well-overlapped with the red fluorescence signal from Mito-Tracker Deep Red with
the Pearson’s correlation coefficient being 0.997. In comparison, the Pearson’s
correlation coefficients were determined as 0.548 and 0.377 with ER-Tracker Red and
Lyso-Tracker Red, respectively (Figure S6). These results indicate clearly that after
activation PDHH is selectively localized in mitochondria, known as the major site of
cellular ROS production and regulators of apoptosis. Similar mitochondrial
localization was also observed in its parent catechol DHH (Figure S7). Consequently
there are two possibilities of either activation of PDHH to DHH by cytoplasmic GSH
followed by localization of DHH to mitochondria or localization of PDHH to
mitochondria followed by its activation to DHH by mitochondrial GSH. Additionally,
the mitochondrial localization was also observed in the case of PDHB, PDHS and
their corresponding catechols (DHB and DHS), even in the case of DPHE (Scheme 2)
bearing no any phenolic hydroxyl group (Figure S7). To our knowledge, this is the
first report of the diphenylpolyene skeletons capable of selectively targeting
mitochondria without the need of additional introducing mitochondria-directed groups
such as the widely used triphenylphosphonium [48-51].
Figure 6 here
3.7. Visualization for therapeutic efficacy of PDHH
2
1

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
PDHH was further used to monitor SW620 cell death together with propidium
iodide (PI), a widely used nuclear fluorescent dye for late apoptotic cells. To induce
rapid cell death, SW620 cells were exposed to 15 ꢀM PDHH for different time points.
As the incubation time increased, PI entered gradually the cells and stained the nuclei,
resulting in a strong red fluorescence signal in the nuclei region, which is indicative of
the occurrence of late cell apoptosis (Figure 7). In contrast, after treatment with
PDHH for 24 h, the cells demonstrated additional typical characteristics of cell
apoptosis including shrinkage of cytoplasm along with formation of vacuoles and
apoptotic bodies (Figure 7), highlighting its superiority in monitoring late cell
apoptosis.
Figure 7 here
3.8. Prooxidative role of PDHH
Having established the theranostic role of PDHH for its release, location and
therapeutic efficacy, we turned our attention to clarify whether its prooxidative role is
responsible for its ability to preferentially kill SW620 cells over L-02 cells. It is
known that under aerobic conditions, catechols are usually unstable and easily
converted into its o-quinone intermediates via two-electron oxidation along with
generation of ROS from the oxidation [23-29] (see also Figure 8A). Additionally, the
o-quinone intermediates are the Michael acceptor-type electrophiles, which could be
conjugated with intracellular nucleophilic GSH, leading to depletion of GSH and
intracellular generation of ROS [52] (see also Figure 8A). Therefore, ROS derived
2
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
from the oxidation of catechols mediate usually their anticancer activity [23-29].
The involvement of ROS (mainly including H O ) in the cytotoxicity of PDHH
2
2
against SW620 cells is supported from significant cytotoxic revision (Figure 8B) by
DTT (a sulfhydryl-containing nucleophile capable of quenching electrophic o-quinone
and ROS) and CAT (a specific scavenger of H O ). To clarify the production of the
2
2
o-quinone of DHH and its quenching by DTT, we investigated its Cu(II)-induced
UV/Vis absorption changes under aerobic conditions. Addition of Cu(II) to DHH in
CH CN/water (1/1, v/v) caused a rapid decrease of its absorption peak centered at 362
3
nm along with an immediate appearance of a new band in the range of 400-700 nm
due to the production of the o-quinone of DHH (Figure 8C). The new band decreased
promptly with the time extended, implying that the o-quinone of DHH is very labile
(the inset in Figure 8C). Meanwhile, the new band can be smoothened by adding DTT,
supporting strongly the quenching of the o-quinone (Figure 8C).
The cytotoxic revision by CAT hints at the role H O in the cytotoxicity of PDHH,
2
2
we thus used conjunctively a fluorescence probe DHE and CAT to imagine the
intracellular generation of H O in living SW620 cells. The probe emits red
2
2
fluorescence upon its reaction with superoxide and other oxidants (e.g., iron and H O ,
2
2
cytochrome c and H O ) [53, 54]. Upon treament of SW620 cells with PDHH for 6 or
2
2
9 h, the red fluorescence appeared in SW620 cells, but not in L-02 cells, and the
increased red fluorescence in SW620 cells was significantly revised by CAT (Figure
8D). Noticeably, SW620 cells were almost intact based on the treatment with PDHH
for 9 h (Figure S8), thereby ruling out the possibility that cytochrome c released from
2
3

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
mitochondria into cytosol would catalyze the reaction of DHE with H O . The above
2
2
results correlated well with the reversion of CAT on the cytotoxicity (Figure 8B) to
further support the intracellular generation of H O in SW620 cells treated with
2
2
PDHH. It should be pointed out that CAT is not membrane-permeable due to its large
molecular size, but as a neutral small molecule H O can diffuse across cell
2
2
membranes [55] and CAT retained extracellularly can scavenge H O diffused from
2
2
inside cells to reduce intracellular H O and to thereby revise the cytotoxicity of
2
2
PDHH. This also indicates that significant amounts of H O leaks into the culture
2
2
media. Therefore, we further carried out the Amplex Red assay [44] to clarify this
point. As illustrated in 8E, treatment with PDHH induced dose- and time-dependently
the H O release from SW620 cells, which was significantly inhibited by CAT,
2
2
providing a direct proof for the generation of H O induced by PDHH.
2
2
The inceased H O was also associated with dose- and time-dependent decrease of
2
2
GSH (Figure 8F) (another important indicator of redox status), suggesting that PDHH
induces redox imbalance of SW620 cells. To identify whether PDHH induces collapse
of mitochondrial membrane potential of SW620 cells, we subsequently performed the
rhodamine 123 experiment in combination with using FCCP (a mitochondrial
uncoupler) as the control [56]. As shown in Figure 9A, treatment with either FCCP or
PDHH decreased the fluorescence intensity of rhodamine 123. The above results
demonstrate that the probe works in the normal and not in the quenching (increased
fluorescence) mode [56], and PDHH induces collapse of mitochondrial membrane
potential of SW620 cells.
2
4

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
The redox imbalance and subsequent collapse of mitochondrial membrane potential
facilitated final apoptosis of SW620 cells (Figure 10A). This was supported by
reversion of CAT and DTT on redox imbalance such as accumulation of H O (Figure
2
2
8D and E) and depletion of GSH (Figure 8F), collapse of mitochondrial membrane
potential (Figure 9A) and apoptosis (Figure 10A) of SW620 cells, induced by PDHH.
In a sharp contrast, PDHH failed to induce the redox imbalance (Figure 8D and F),
collapse of mitochondrial membrane potential (Figure 9B) and apoptosis in L-02 cells
(Figure 10B). The above results provide an entire evidence chain to support that
PDHH works as a PAA after its activation to DHH by GSH in SW620 cells.
Figures 8-10 here
In summary, we used the GSH-mediated conversion of DNBS to phenols to protect
a catechol moiety and designed stable pro-catechol-type diphenylpolyenes as small
molecule-based anticancer theranostic prodrugs. The designed molecules were
synthesized via a modular route allowing creation of various pro-catechol-type
diphenylpolyenes and introduction of other protecting group onto the catecholic 4-OH.
Dissimilar to the currently available theranostic prodrugs, the designed PDHH is a
concise molecule permitting a turn-on fluorescent monitoring for its release, targeting
mitochondria and therapeutic efficacy. As a potent PAA, PDHH is capable of
exploiting increased levels of GSH in cancer cells to in situ release a catechol moiety
followed by its in situ oxidation to toxic o-quinone, leading to preferential redox
imbalance (including depletion of GSH and generation of H O ) and final selective
2
2
2
5

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
apoptosis of cancer cells over normal cells. It is also superior to 5-fluorouracil and
doxorubicin, the widely used chemotherapy drugs, in terms of its ability to kill
preferentially SW620 over normal L-02 cells. Additionally, we identified the
diphenylpolyene skeletons as novel mitochondria-directed groups that should prove
useful for creating new mitochondria-targeted fluorescence probes and drugs.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(Grant No. 21672091)
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version
at http://dx.doi.org/10.1016/j.freeradbiomed.
References
2
6

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
[1].
E. S. Andersen, M. Dong, M. M. Nielsen, K. Jahn, R. Subramani, W.
Mamdouh, M. M. Golas, B. Sander, H. Stark, C. L. P. Oliveira, J. S. Pedersen,
V. Birkedal, F. Besenbacher, K. V. Gothelf, J. Kjems, Self-assembly of a
nanoscale DNA box with a controllable lid, Nature 459 (2009) 73-76.
M. H. Lee, A. Sharma, M. J. Chang, J. Lee, S. Son, J. L. Sessler, C. Kang, J. S.
Kim, Fluorogenic reaction-based prodrug conjugates as targeted cancer
theranostics, Chem. Soc. Rev. 47 (2018) 28-52.
[2].
[3].
[4].
J.-Z. Du, X.-J. Du, C.-Q. Mao, J. Wang, Tailor-made dual pH-sensitive
polymer-doxorubicin nanoparticles for efficient anticancer drug delivery, J.
Am. Chem. Soc. 133 (2011) 17560-17563.
J. L. Major Jourden, S. M. Cohen, Hydrogen peroxide activated matrix
metalloproteinase inhibitors: a prodrug approach, Angew. Chem. Int. Ed. 49
(2010) 6795-6797.
[5]. Y. Kuang, K. Balakrishnan, V. Gandhi, X. Peng, Hydrogen peroxide inducible
DNA cross-linking agents: targeted anticancer prodrugs, J. Am. Chem. Soc. 133
(2011) 19278-19281.
[6].
E.-J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K. S. Hong, J.
S. Kim, An activatable prodrug for the treatment of metastatic tumors, J. Am.
Chem. Soc. 136 (2014) 13888-13894.
[7].
R. Kumar, J. Han, H.-J. Lim, W. X. Ren, J.-Y. Lim, J.-H. Kim, J. S. Kim,
Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor
theranostic prodrug: in vivo imaging and precise cancer treatment, J. Am.
2
7

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
Chem. Soc. 136 (2014) 17836-17843.
[8].
[9].
M. Ye, X. Wang, J. Tang, Z. Guo, Y. Shen, H. Tian, W.-H. Zhu, Dual-channel
NIR activatable theranostic prodrug for in vivo spatiotemporal tracking
thiol-triggered chemotherapy, Chem. Sci. 7 (2016) 4958-4965.
F. Kong, Z. Liang, D. Luan, X. Liu, K. Xu, B. Tang, A glutathione
(GSH)-responsive near-infrared (NIR) theranostic prodrug for cancer therapy
and imaging, Anal. Chem. 88 (2016) 6450-6456.
[10]. X. Wu, X. Sun, Z. Guo, J. Tang, Y. Shen, T. D. James, H. Tian, W. Zhu, In
vivo and in situ tracking cancer chemotherapy by highly photostable NIR
fluorescent theranostic prodrug, J. Am. Chem. Soc. 136 (2014) 3579-3588.
[11]. S. Bhuniya, S. Maiti, E.-J. Kim, H. Lee, J. L. Sessler, K. S. Hong, J. S. Kim,
An activatable theranostic for targeted cancer therapy and imaging, Angew.
Chem. Int. Ed. 53 (2014) 4469-4474.
[12]. S. Santra, C. Kaittanis, O. J. Santiesteban, J. M. Perez, Cell-specific,
activatable, and theranostic prodrug for dual-targeted cancer imaging and
therapy, J. Am. Chem. Soc. 133 (2011) 16680-16688.
[13]. Y. Ichikawa, M. Kamiya, F. Obata, M. Miura, T. Terai, T. Komatsu, T. Ueno, K.
Hanaoka,
T.
Nagano,
Y.
Urano,
Selective
ablation
of
β-galactosidase-expressing cells with a rationally designed activatable
photosensitizer, Angew. Chem. Int. Ed. 53 (2014) 6772-6775.
[14]. G. Zheng, J. Chen, K. Stefflova, M. Jarvi, H. Li, B. C. Wilson, Photodynamic
molecular beacon as an activatable photosensitizer based on
2
8