Enhanced reactive oxygen species levels by an active benzothiazole complex-mediated fenton reaction for highly effective antitumor therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00819

Breaking the threshold of intracellular reactive oxygen species (ROS) levels can cause nonspecific oxidative damage to proteins and lead to the Fenton reaction-mediated exogenous ROS production to be a new promising anticancer strategy. However, the problems, including the inefficient transport of metal catalysts and insufficient endogenous hydrogen peroxide (H₂O₂) content in cells, still need to be improved. In this study, a functional nanosystem encapsulated with benzothiazole complexes (FeTB₂) and the photosensitizer indocyanine green (ICG) was designed for highly effective antitumor therapy. The surface of the nanocarriers was modified with dihydroartemisinin (DHA)-grafted polyglutamic acid. The induced hyperthermia enables the lipid-polymer shell to depolymerize, releasing FeTB₂. The released FeTB₂ could kill tumor cells in two different ways by inhibiting DNA replication and catalyzing H₂O₂ to produce active a OH. Moreover, the conjugated DHA could increase the amount of peroxides in tumor cells and significantly enhance the ROS yield. This work has provided solid evidence that the present nanosystem enables a significant effect on tumor killing through the combined inhibition of DNA replication and ROS-mediated oxidative damage by regulation of the tumor microenvironment, providing a ROS-mediated high-efficiency antitumor strategy.

Full text of DOI:10.1021/acs.molpharmaceut.9b00819

Subscriber access provided by UB der LMU Muenchen
Article
Enhanced Reactive Oxygen Species Levels by Active Benzothiazole
Complex mediated Fenton Reaction for Highly Effective Anti-tumor Therapy
Chaoqun You, Hongshuai Wu, Zhiguo Gao, Fanghui Chen,
Like Ning, Yu Zhang, yixin Dong, Baiwang Sun, and Fei Wang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00819 • Publication Date (Web): 29 Oct 2019
Downloaded from pubs.acs.org on November 3, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
An active benzothiazole complex-filled Fenton reaction activable FeTB2@DHA−INPs polymer grafted
liposomes to display ROS mediated chemodynamic therapy (CDT).
744x515mm (72 x 72 DPI)
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 28
1
2
3
4
5
6
7
8
Enhanced Reactive Oxygen Species Levels by
Active Benzothiazole Complex Mediated
Fenton Reaction for Highly Effective Anti-
Tumor Therapy
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chaoqun You 1, Hongshuai Wu 2, Zhiguo Gao 2, Fanghui Chen 2, Like Ning 1, Yu Zhang
1, Yixin Dong 1, Baiwang Sun 2,*, Fei Wang 1,*
1 College of Chemical Engineering, Nanjing Forestry University; Jiangsu Key Lab for
the Chemistry and Utilization of Agro-Forest Biomass, Nanjing 210037, PR China Fax:
+86 25 85427649, Tel: +86 25 85427649, E-mail address: hgwf@njfu.edu.cn
2 School of Chemistry and Chemical Engineering, Southeast University, Nanjing
210089, PR China. Fax: +86 25 52090614, Tel: +86 25 52090614, E-mail address:
chmsunbw@seu.edu.cn
Abstract
Breaking the threshold of intracellular reactive oxygen species (ROS) levels can
cause non-specific oxidative damage to proteins, and lead to the Fenton reaction-
mediated exogenous ROS production to be a new promising anticancer strategy.
However, the problems including the inefficient transport of metal catalysts and
insufficient endogenous hydrogen peroxide (H2O2) content in cells still need to be
improved. In this study, functional nanosystem encapsulated with benzothiazole
complexes (FeTB2) and the photosensitizer indocyanine green (ICG) was designed for
highly effective antitumor therapy. The surface of the nanocarriers was modified with
dihydroartemisinin (DHA)-grafted polyglutamic acid. The induced hyperthermia
enables the lipid-polymer shell to depolymerize, releasing FeTB2. The released FeTB2
could kill tumor cells in two different ways by inhibiting DNA replication and
catalyzing H2O2 to produce active •OH. Moreover, the conjugated DHA could increase
the amount of peroxides in tumor cells and significantly enhance ROS yield. This work
has provided solid evidence that the present nanosystem enables significant effect on
1
ACS Paragon Plus Environment

Page 3 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
tumor killing through the combined inhibition of DNA replication and ROS mediated
oxidative damage by regulation of tumor microenvironment, providing a ROS-
mediated high efficiency antitumor strategy.
9
Keywords: Reactive oxygen species, Dihydroartemisinin, Benzothiazole derivatives,
Fenton reaction, Synergistic therapy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1. Introduction
Reactive oxygen species (ROS) show important biological functions in organisms.
ROS includes superoxide anion radicals (O2•-), singlet oxygen (1O2), hydrogen
peroxide (H2O2) and hydroxyl radicals (•OH).1,2 Different levels of ROS lead to
different biological effects in cells. When intracellular ROS levels are low, it acts as an
important 2nd messenger in cell proliferation and homeostasis by reversibly oxidizing
the mercapto group of proteins to modify their structure and function. For example,
ROS promotes the expression of Cyclin D1, activates MAPK (mitogen-activated
protein kinase), ERK (extracellular signal regulated kinase), JNK (c-Jun N-terminal
kinase), all of which promote cell growth and survival.3 However, when intracellular
ROS levels are high, the tumor suppressor PTEN (phosphatase and tensin homolog
deleted on chromosome ten) is reversibly inactivated,4 leading to the non-specific
oxidative damage of cellular DNA, proteins and lipids, subsequently promoting
apoptosis, necrosis, and cancer.5
Studies have shown that in the presence of Fe2+, H2O2 can be converted to •OH by
the Fenton reaction. Since •OH has a short half-life (10-9 s) and high reactivity, it does
not diffuse from the production site, but rapidly oxidizes surrounding biomolecules.
This new regulatory form of cell death that relies on Fe2+ and ROS is called
Ferroptosis.6-8 Moreover, this treatment by regulating ROS levels in tumor cells,
representing a new anticancer strategy.
When designing the Fenton reaction system, ROS generation is maximized under
the premise of good biocompatibility and lack of toxic side effects, so as to obtain a
high tumor killing efficiency. In order to obtain maximum catalytic efficiency,
transporting metals in the form of ions can effectively improve the load efficiency. Nie
2
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 28
1
2
3
4
5
6
7
8
9
et al reported iron oxide nanoparticles loaded with linoleic acid hydroperoxide
(LAHP).9 The surface of the nanoparticles dissociated Fe2+ and reacted with LAHP to
generate large amount of ROS following their internalization into cancer cells. Shi et al
used endogenous H2O2 in tumor cells, which when combined with Fe2+ and FeS2,
respectively, produced large amount of ROS, leading to high anticancer efficiency.10,11
Benzothiazole contains a benzene ring embedded five-membered heterocyclic ring,
that has been widely used for the research and development of anti-tumor drugs.12,13
Benzothiazole derivatives exert anti-tumor effects through kinase inhibition, apoptosis
induction, and action on DNA.14-17 El-Falouji et al18 reported a series of benzothiazole
complexes in which a benzothiazole derivative and a trivalent metal ion were
coordinated. These complexes exhibited strong binding ability to calf thymus DNA and
showed anti-angiogenic effect.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Artemisinin is a sesquiterpene lactone containing a peroxide group which is
isolated from the plant Artemisia, artesunate, artemether and dihydroartemisinin (DHA)
which are derivatives of artemisinin. Among them, DHA molecules have higher water
solubility, are more easily absorbed, display high efficiency, and low toxicity.19 Recent
studies found that,20-22 DHA acts on the mitochondria-dependent apoptotic pathway to
inhibit NF-κB activation, thereby promoting tumor cell apoptosis and inhibiting cell
cycle progression. Moreover, DHA directly inhibits tumor cells through Fe2+ catalysis.
Finally, DHA acts on the fibrinolytic system uPA and inhibits VEGF-induced
angiogenesis to inhibit tumor invasion and metastasis.
At present work, we designed and prepared a functional nanocarrier that was
internally encapsulated with functional benzothiazole complexes (FeTB2) and the
photosensitizer indocyanine green (ICG). The surface of the nanocarrier was modified
with DHA-grafted polyglutamic acid. The hyperthermia induced by the photothermal
conversion performance of ICG under NIR irradiation caused the lipid-polymer shell
to depolymerize, releasing FeTB2. The released FeTB2 could not only directly killed
tumor cells by inducing DNA arrest, but the Fe3+ catalyzes H2O2 production in the
tumor microenvironment to produce large levels of active •OH, which further leads to
oxidative damage. In addition, the conjugated DHA further complements the
3
ACS Paragon Plus Environment

Page 5 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
intracellular peroxides and affectively enhances the transition of Fenton reactions. The
present nanosystems effectively kill tumor cells by inducing DNA arrest and ROS
mediated oxidative damage, and providing a ROS-mediated high-efficiency antitumor
strategy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2. Materials and Methods
2.1 Materials
2-Mercaptobenzothiazole,
Dihydroartemisinin,
Cholesterol,
Lecithin,
Indocyanine green (ICG) were purchased from Sigma-Aldrich Chemical Company (St.
Louis, MO, USA). Poly-L-glutamic acid, HOOC-PEG2000-DSPE was purchased from
Polymtek Biomaterial Co., Ltd. (Shenzhen, China). All the dye kits were obtained from
Beyotime Biotechnology Co., Ltd (Shanghai, China). All the other chemical reagents
were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China).
2.2 Synthesized Procedure
2.2.1 Synthesis and characterization of Fe(III) -2- thioacetate benzothiazole
The 2-Thioacetic acid benzothiazole was prepared according to the method in
literature.23 Briefly, 2-Mercaptobenzothiazole (0.90 g, 3.0 mmol), Chloroacetic acid
(1.15 g, 6.0 mmol) were dissolved in aqueous methanol (20 mL) in the presence of
KOH (0.1 mol) and the mixture was refluxed for 4 h. Then the mixture solution was
filtered and washed with diluted hydrochloric acid (3×5 mL), extracted with aqueous
ethanol (3×10 mL). The details of the synthetic procedure was shown in Figure S1.
The desired intermediate products were obtained after being concentrated and dried
under reduced pressure. The product was recovered as a yellowish white solid (0.65 g,
73%). 1H NMR (600 MHz, DMSO-d6): d (ppm) 11.12 (d, 1H), 7.48 (d, 1H), 7.36 (d,
1H), 7.25-7.30 (d, 2H), 1.73 (m, 2H); ESI-MS (m/z): 226.1312 [M + H]+.
Next, 2-Thioacetic acid benzothiazole (1.0 mmol, 0.224 g), triethylamine (1.0
mmol, 0.102 g) was dissolved in ethanol (20 mL) and the mixture was stirred for 20
min at 25 oC. The FeCl3.6H2O dissolved in ethanol (0.5 mmol, 20 mL) and then added
slowly to the prepared solution. The brown precipitate appeared immediately after
mixing the two components and then stirred overnight at room temperature. The
4
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 28
1
2
3
4
5
6
7
8
9
product was obtained after filtration, purification and drying under vacuum. The desired
product as a brown solid (0.79 g, 55%). Yield: 55%; IR (KBr, n/cm-1): 3342, 1730, 1703,
1621, 1556, 1026, 686 and 513 cm-1; 1H NMR (600 MHz, DMSO-d6): d (ppm) 7.73 (d,
1H), 7.41 (d, 1H), 7.25–7.30 (d, 2H), 2.52 (m, 2H); ESI-MS (m/z): 504.8122 [M + H]+.
2.2.2 Synthesis and characterization of PGlu-SS-DHA lipid-polymer chains (DHA-
PGlu–PEG2000–DSPE)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The dihydroartemisinin modified lipid-polymer chains was synthesized through
poly-L-glutamic acid with three steps as described in Figure S2. The intermediate
products (PGlu-PEG2000-DSPE and DHA-PIP) were synthesized according our
previous study.24 Next, PGlu-PEG2000-DSPE (0.50 g, 0.12 mmol) and DHA-PIP (0.13
g, 0.36 mmol) were dissolved in DMF (15 mL) and then the mixture was stirred for 24
h at room temperature in the presence of DMAP (0.02 g, 0.15 mmol). Then the white
precipitate formed as the reaction progressed and then transferred, redissolved in
deionized water (5 mL). The desired product was freeze-dried from deionized water
with a solid powder form after dialysis (MWCO 4000Da, water was replaced every 6
h).1H NMR (600 MHz, DMSO-d6): d (ppm) 5.34 (d, 10H), 4.05 (d, 10H), 4.01 (d, 10H),
3.36 (d, ~10H), 3.05–2.51 (b, ~4000H), 1.99–0.75 (m, ~400H).
2.3 Self-Assembly and Characterizations
The designed functional nanosystems (FeTB2@DHA-INPs) were prepared
according the published literatures.25-27 Briefly, DHA-PGlu10–PEG2000–DSPE (3.0 mg),
cholesterol (2.0 mg) and lecithin (20.0 mg) were dissolved in chloroform (40 mL) and
a thin film was formed after solvent evaporation. To this film was added 15 mL of
FeTB2 aqueous solution (0.2 mg mL-1), 5 mL of ICG aqueous solution (0.4 mg mL-1)
and then ultrasonic vibrated for 10 min. The prepared nanosystem suspension was then
centrifuged at 2500 rpm for 20 min, the supernatant was then collected, filtrated and
lyophilized.
2.4 Photothermal Effects and Drug Release
To evaluate the photothermal effects of the prepared nanosystem, different
concentrations of FeTB2@DHA-INPs suspension (10, 20, and 40 μg mL-1) were
investigated after irradiation with a near-infrared laser (808 nm, 1.5 W cm-1) for 5 min
5
ACS Paragon Plus Environment

Page 7 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
and the temperature variation was detected with an infrared (IR) thermal camera (every
30 s). Before the evaluation, the photostability of FeTB2@DHA-INPs suspension were
tested.
9
The in vitro drug release of FeTB2@DHA-INPs was performed as the published
literatures described.28-30 Briefly, a predesigned amount of the suspensions (3 mL, 1.5
mg mL -1) in dialysis bag (MW, 10 000) was immersed in PBS (15 mL, pH = 7.4, 6.8
and 5.5) and then irradiated by an 808 nm near-infrared laser (1.5 W cm-1, 5 min). A
certain volume (2 mL) of the dissolution medium was taken out during each designed
release period and an equal volume of fresh medium was added instead. The amount of
released FeTB2 in the dialysate was calculated by ICP-AES and each group was tested
in triplicate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.5 Uptake and Co-localization into Mitochondria.
A549 and MCF-7 cells were cultured in 6-well plates (9.0×104 cells well-1) and
cultured for 24 h, then a certain concentration of ICG (50.0 μg mL-1) in FeTB2@DHA-
INPs suspension (3 mL) was added into the medium. The cells were cleaned and stained
with DAPI and further observed by CLSM after been incubated for 1.5, and 3.0 h. In
addition, the fluorescence intensity of the intracellular ICG was calculated by flow
cytometer. Next, the cells were treated with FeTB2@DHA-INPs as above and then
incubated for 3 h, stained with Lysotracker Green (lysosome staining, green color) to
evaluate the uptake of lysosomes.
2.6 Reactive Oxygen Species (ROS) Generation
A549 and MCF-7 cells were seeded in 6-well plates at a density of 3.0 ×105 cells
per well and cultured for 24 h. The cells were then treated with different concentrations
(10, 20, and 30 μg mL-1) of nanoparticles for 3 h. Then the cells were treated with DCF-
DA (10 µM) for 30 min and then analysis by flow cytometry. Similarly, different
incubation time (0.5 h, 1.0 h, 1.5 h, 3.0 h) and different nanoparticles formulations
(Control group, free TeTB2, FeTB2@INPs, FeTB2@DHA-INPs, and FeTB2@DHA-
INPs+NIR) were further investigated, respectively. In addition, the predesigned
concentrations of all formulations were 10 µM.
2.7 In Vitro Cytotoxicity
6
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 28
1
2
3
4
5
6
7
8
9
A549 and MCF-7 cells were seeded in 96-well plates (7×103 cells well-1) and
incubated overnight until fully covered. The cells were then treated with various
formulations of nanoparticles at certain concentrations (0.1, 2.5, 5.0, 7.5, 10.0 μg mL-
1). After 12 h of incubation, the medium was replaced and the wells were irradiated
with a NIR laser (808 nm, 1.5 W cm-1, 3 s well-1) and further incubated for another 12
h. Next, the standard MTT assay was performed to evaluate the inhibition effect of
FeTB2@DHA-INPs. Moreover, we take the same approach to evaluate the cytotoxicity
of the present nanoparticles on normal cells (RGC-5), and the effect of different DHA
concentrations (0.10, 0.75, 1.50, 3.00, 6.00 μg mL-1) on the toxicity of the nanoparticles
to MCF-7 cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.8 Cell Apoptosis and Cycle Arrest
To further evaluate the induced apoptosis effect of FeTB2@DHA-INPs on tumor
cells, MCF-7 cells were incubated in 6-well plates (5.0×105 cells per well) for 24 h until
the cells were fully covered. Then the medium were replaced with blank nanoparticles,
FeTB2@INPs, and FeTB2@DHA-INPs for 12 h, and then treated with NIR laser for 3
min. After been incubated for another 12 h, the cells were collected (1000 rpm, 5 min,
4 °C) and washed with cold PBS and then suspended in 500 µL binding buffer. Next, 5
µL Annexin V-FITC and 10 µL PI was added to the cell suspension, incubated for 20
min in dark and analyzed by flow cytometry. The concentration of each sample was set
as 5 µM.
Next, the cell cycle detection was performed using the same cell processing
procedure. Specially, after been incubated with free FeTB2 and FeTB2@DHA-INPs for
48 h, the collected cell suspensions were fixed with 70% ethanol overnight at 4 oC, and
further treated with DNA staining for 30 min in the dark. The final cell samples were
analyzed with a BD Accuri C6 Flow Cytometer.
2.9 In Vivo Imaging and Biodistribution Analysis
The FeTB2@DHA-INPs solutions containing 45 μg mL-1 ICG were intratumorally
injected into the nude mice, then in vivo imaging and FL quantitative analysis of ICG
(excitation/emission wavelength: 704/735 nm) by the ex/in vivo imaging system (CRI
maestro, USA) at 0, 3, 6, 12, and 24 h was performed after injection. In addition, the
7
ACS Paragon Plus Environment

Page 9 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
mice were sacrificed after 24 h of injection, then the tumor and major organs including
the heart, liver, spleen, lung and kidney were collected for imaging and quantitative
biodistribution analysis.
9
2.10 In Vivo Antitumor Efficacy Studies Using a Breast Cancer Model
Male BALB/c mice bearing MCF-7 tumors (~100 mm3) were randomly divided
into five groups (6 per group), and intratumorally administered 150 μL of free FeTB2
or FeTB2@DHA-INPs solution at a concentration of 16.67 mg kg-1 (containing 1.64
mg of FeTB2 and 0.11 mg of DHA) and the experimental group was further irradiated
with a NIR laser (808 nm, 5 min). Then the mice were treated every 3 days for a total
of 5 treatments. The mice were observed for total 24 days and the tumor sizes were
measured by an electronic digital caliper every 3 days after treatment and calculated as
follows: Tumor volume (V) = (L×W2)/2, in which the length (L) is the longest part, and
the width (W) is the narrowest section perpendicular to the length. After sacrifice, the
tumors, livers and kidneys after treatment for 24 days were observed by H&E staining.
2.11 Statistical Analysis Data are expressed as mean ± SD. The differences among
groups were determined using one-way ANOVA analysis followed by Tukey’s post-
test; a P value of < 0.05 was considered significant. Statistical significance was defined
as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. P > 0.05, no significance.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Results and Discussion
3.1 Synthesis, Assembly and Characterization
In order to effectively increase the efficiency of Fenton reaction, the Fe3+-loaded
coordination compound (FeTB2) and the functionalized polymer conjugated with DHA
(DHA-PGlu10-PEG-DSPE) were designed and synthesized (Figure S1, S2). All the
experimental steps were carried out under mild conditions and the yields of obtained
products were satisfactory. The chemical structures of the intermediates and final
1
products were confirmed by commonly used spectroscopic techniques including H
NMR and ESI-MS, and the results are shown in Figure S3-S5. The assignment of the
protons in the molecular structure of the compounds and the molecular weight data
were listed in the experimental section. In addition, the molecular weight of functional
8
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 28
1
2
3
4
5
6
7
8
9
polymers was characterized by GPC as shown in Table S1. The results showed that the
experimental data were consistent with the standard data of the targeted molecules.
The FeTB2@DHA-INPs were prepared using the classic film hydration method as
shown in Scheme 1. In order to maximize the loading capacity of the drugs, different
composition proportions of FeTB2, DHA-PGlu10-PEG-DSPE, and Lecithin were tested,
the results exhibited that the amount of loaded FeTB2 reach 9.86% at the ratio of 5:3:20
(Table S2). In addition, the particle size of this self-assembling formula was acceptable
and showed an average particle size of 122.4 nm with polydispersity of 0.212 while
those without polymer ligands were about 105.7 nm (Figure 1A). According to the
existing reports,31 this size specification could ensure the particles are not be cleared
during blood circulation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, UV-vis spectroscopy was employed to character the drug loaded
nanoparticles. The spectrum showed the maximum characteristic absorbance for FeTB2
at 320 nm while DHA did not show obvious absorption (Figure 1B). Moreover, the
FeTB2@DHA-INPs showed maximum characteristic absorption peak around 280 nm
in the UV spectrum. The morphological characteristics of the prepared FeTB2@DHA-
INPs observed by TEM are shown in Figure 1C, and the results showed that the
currently nanoparticles process good monodispersity and regular spherical structure.
The depolymerized form of nanoparticles after been exposed to NIR light indicated the
present nanoparticles showed excellent stimulus-response capacity (Figure 1D). The
zeta potential results of the nanoparticles showed that the FeTB2@DHA-INPs was
positively charged (23.93 mV) due to conjugated DHA while it was −23.93 mV without
conjugation (Figure 1E). When the surface of the positively charged lipid layer is
conjugated with the DHA ligands, the surface charge of the nanoparticles could become
negative to balance the tension in the aqueous solution to keep monodispersity. Figure
1F showed that different types of nanoparticles present different color states.
3.2 Photothermal Conversion and Stimulus-Response Release
In order to evaluate whether the heat generated by photothermal conversion of the
nanoparticles is sufficient to cause the depolymerization of the lipid-polymer shell, the
induced heat was recorded and showed in Figure 2A. As exhibited, the control groups
9
ACS Paragon Plus Environment

Page 11 of 28
Molecular Pharmaceutics
1
2
3
4
(PBS or hollow nanoparticles) did not show obvious temperature change and the
5
6
o
recorded temperature was only about 25 C. However, the induced temperature of
7
8
9
nanoparticles with ICG encapsulated groups showed the regular of concentration-
dependent temperature increase. The induced hyperthermia can increase to about 55 oC
after been exposed to NIR laser for 5 min (20 μg mL-1) and which has exceeded the
phase transition temperature of the lipid-polymer shell.32,33 In addition, the highest
induced temperature was about 59 oC after been exposed to the NIR laser for 5 min and
which can even lead to tumor ablation. Figure 2B showed the morphology of the
nanoparticles solution by thermal imaging after been irradiated by NIR laser. Thus,
based on the NIR irradiation, the present nanoparticles are sufficient to act as a role for
controlled drug release or even for photothermal therapy of tumors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, we calculated the amount of the released drug from the nanoparticles under
different conditions using the dialysis method. Considering the fact that the lipid-
polymer shell of the current nanoparticles can be destroyed by acidic environment, we
used three different pH values (7.4, 6.8, or 5.5) to explore the drug release behavior
(Figure 2C). Overall, the amounts of released FeTB2 increased more significantly after
been exposed to NIR laser as compared with the groups without NIR irradiation.
Specially, the amounts of the released FeTB2 increased slowly after been releasing for
more than 48 h at pH=7.4. However, the release of FeTB2 was observed with a
cumulative drug release of 31.62% after NIR irradiation at pH=7.4 for 48 h. It is worth
noting that FeTB2@DHA-INPs released <20% of drug molecules in the absence of NIR
irradiation at pH=7.4 and this reduction ratio will become higher in lower pH value.
This release behavior indicates that FeTB2@DHA-INPs are highly responsive to NIR
light and acidic environment. Figure 2D showed the depolymerization status of the
nanoparticles after been released by NIR irradiation at pH=7.4 for 48 h.
3.3 Cellular Uptake and Intracellular ROS Production
The cellular uptake was observed by CLSM and the results are shown in Figure
3A. Only weak red fluorescence intensity (tracing the FeTB2@DHA-INPs) was
appeared in A549 cells after been incubated for 1.5 h when compared with it in MCF-
7 cells. However, the red fluorescence intensity increased obviously in the cytoplasm
10
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 28
1
2
3
4
5
6
7
8
9
as the incubation time was extended to 3 h in both A549 and MCF-7 cells. Next, we
quantified the fluorescence intensity using a flow cytometer (Figure 3B) and further
made a comparison (Figure 3C). The quantitative results indicated the amount of
accumulated fluorescence intensity in both tumor cells were significant difference after
3 h of incubation (*P<0.05). Thus, the obtained results indicated that the cellular uptake
of FeTB2@DHA-INPs present a time-dependent pattern, providing the basis for the
study of intracellular ROS levels. Before the ROS evaluation, we use Mito-Tracker
Green (MTG) to label mitochondria for the imaging observation. As exhibited in Figure
3D, the obviously strong fluorescence signals were found in both A549 and MCF-7
cells after been incubated for 3 h.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, the induced ROS was calculated using the 2’, 7’-dichlorodihydrofluorescein
diacetate probe (DCFH-DA) and CLSM was used to monitor intracellular ROS
production. The free ICG were incubated with MCF-7 cells for 12 h as the control
groups. As exhibited in Figure 4A, the amount of ROS generation increased with the
concentration of nanoparticles solution. Moreover, the amount of generated ROS in the
mitochondria when incubated with FeTB2@DHA-INPs at the concentration of 30 μg
mL-1 was 7.72-fold than that at 10 μg mL-1 (**P<0.01, Figure 4D). Likewise, the
production of ROS increased with the increase of incubation time and the amount of
generated ROS for 3 h of incubation was much higher than that only incubated for 1 h
(***p<0.001, Figure 4B, 4E).
It is important to note that the free ICG also can lead to the increase of intracellular
ROS levels. This is because ICG could cause cell apoptosis as a drug molecule and
induced ROS generation. In contrast, the FeTB2@DHA-INPs treated group
demonstrated much higher levels of ROS generation when compared with free ICG
group (Figure 4C, 4F). This may attribute to oxidative stress from the released FeTB2
that is capable of promoting the parent drug release, and ultimately increase in the ROS
levels. Moreover, the conjugated DHA further enhanced this generation efficiency.
Next, in a parallel experiment we further quantitatively evaluated the ROS levels in
A549 cells by flow cytometry after been treated with FeTB2@DHA-INPs (Figure S6).
The obtained results showed that the amount of intracellular drugs were about the same
11
ACS Paragon Plus Environment

Page 13 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
as the results of the fluorescence intensity in MCF-7 cells. Therefore, FeTB2@DHA-
INPs exhibited effective antitumor activity in vitro by generating ROS in cancer cells
3.4 Cytotoxicity and Apoptosis Evaluation
9
Excellent cytotoxicity is the primary characteristic that a new drug molecule is
designed to acquire. Thus, the IC50 (mmolL-1) values in two different tumor cell lines
were investigated (Table S3). As expected, the designed FeTB2 showed favored toxicity
to selected cell lines after been coordinated with Fe3+. Next, the standard MTT assay
was carried out to investigate the in vitro cytotoxicity of FeTB2@DHA-INPs.
As exhibited in Figure 5A and 5B, in general, the cell viability decreased rapidly
with increased concentrations of FeTB2@DHA-INPs and which showed superior
antitumor properties in both A549 and MCF-7 cells. Compared with the results in A549
cells, free FeTB2 showed much higher cytotoxicity in MCF-7 cells after been incubated
for 24 h. However, the free FeTB2 showed lower cytotoxicity at each concentration
when compared with FeTB2@DHA-INPs, which attributed to the addition of DHA
(Figure 5A, 5B). Undoubtedly, FeTB2@DHA-INPs significantly increased ROS levels
through the release of DHA in cancer cells over time. Unlike the chemical drug
molecules, FeTB2@DHA-INPs showed much lower cytotoxicity towards the RGC-5
cells as compared with MCF-7 cells (Figure 5C). In addition, the loaded DHA was
obviously enhanced the cytotoxicity at each dose concentration (Figure 5D). Thus, the
antiproliferative activity of FeTB2@DHA-INPs against RGC-5 is extremely lower may
due to the relatively low ROS levels in normal cells. Finally, due to the perfect
antitumor performance, FeTB2@DHA-INPs should be considered in the application of
cancer therapy for a ROS mediated newly anticancer strategy, together with the
enhanced effect of DHA and NIR-response release behavior to minimize the adverse
effects of chemotherapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The oxidative damage performance of FeTB2@DHA-INPs on MCF-7 cells was
further quantified by flow cytometry. As shown in Figure 5E, the MCF-7 cells treated
with blank nanoparticles showed much lower apoptotic ratio (11.7%), which indicates
the cytotoxicity is mainly caused by the encapsulated FeTB2, while the surface-loaded
DHA cannot effectively kill tumor cells as drug molecules. In addition, the apoptosis
12
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 28
1
2
3
4
5
6
7
8
9
rate increased to 23.6% when the cells incubated with FeTB2 loaded nanoparticles
(FeTB2@INPs), which is about 13-fold to the control group (2.7%). The apoptosis rate
reached to 35.8% when the cells incubated with FeTB2@DHA-INPs for 24 h. As
exhibited above, the ROS generated by catalyzing the intracellular hydrogen peroxid
through the encapsulated FeTB2, and the surface-loaded DHA further increases ROS
levels by supplementing the total peroxide content. Thus, the present nanoparticles
demonstrated strong oxidative damage effect on MCF-7 cells and further suggested that
the prepared nano-agents showed great potential in cancer therapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As shown in Figure 5F and 5G, FeTB2 obviously acts on the G2/M phase of the
cell cycle. Specially, the cell percentage in the S phase increases from 37.02% to
42.73%, while which decrease from 57.24% to 34.86% in the G0/G1 phase, this is
highly consistent with our previous study that benzothiazole derivatives act as a DNA
binding agent to prevent DNA replication.34 In addition, the results further show that
FeTB2@DHA-INPs also interferes with the cell cycle in the G2/M phase. In particularly,
the cell percentage in the G0/G1 phase decreased from 57.24% to 44.98% as well. The
results indicate that FeTB2 remarkably inhibits DNA replication in the G2/M phase.
Based on the obtained results, the proposed mechanism of FeTB2 molecules on
tumor cells is as follows: as FeTB2 is released from FeTB2@DHA-INPs in the tumor
cells, it can not only directly kill tumor cells by inhibiting DNA replication, but the
coordinated Fe3+ is fully released in tumor microenvironment and strongly generated
•OH by catalyzes endogenous H2O2 to cause oxidative damage. Thus it shows the
favorable synergistic effect from the FeTB2 molecules and provides a ROS-mediated
high efficiency antitumor strategy.
3.5 In Vivo Imaging Study
In order to observe the delivery of FeTB2@DHA-INPs in vivo, ICG loaded
FeTB2@DHA-INPs were injected intratumorally into BALB/c mice bearing MCF-7
tumors (n=3). As shown in Figure 6A, in the initial 3 h after injection, the nanoparticles
did not spread obviously and were mainly accumulated in the tumor site. However, the
nanoparticles were gradually spread to the whole body along with blood circulation and
the fluorescence signals were significant in the organs (such as liver and kidneys) at 12
13
ACS Paragon Plus Environment

Page 15 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
h post-injection. It is worth mentioning that the fluorescence signals in tumor sites still
at high levels at 24 h post-injection. Therefore, the obtained results exhibited excellent
endocytosis and accumulation performance of FeTB2@DHA-INPs in tumor sites with
the effect of the lipid-polymer shells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, we deeply evaluated the fluorescence signals intensity accumulated in tumor
and various organs after 24 h of post-injection, and the results exhibited that significant
fluorescence signals were accumulated in the organs (Figure 6B). These results of ICG
imaging in vivo further exhibited that the small therapeutic molecules can be effectively
prevented from being cleared by the lipid–polymer structure in the living body.
3.6 In Vivo Antitumor Therapy
We further applied the FeTB2@DHA-INPs to BALB/c mice to evaluate the
antitumor effect in vivo. As shown in Figure 6C, different from other experimental
groups, the tumor volumes in the control group increased by approximately 3.3-fold in
6 days, while the experimental groups showed a moderate increase due to the obvious
antineoplastic efficacy. In addition, during the period of 24 days treatment, the tumor
volumes in group b, c, d increased by approximately 20.0-fold, 16.8-fold, and 12.6-fold
respectively, while the FeTB2@DHA-INPs added NIR irradiation group (group e)
showed an insignificant tumor volume increase (only 5.0-fold).
Moreover, the body weight in the experimental group (group e) increased slightly
while it in group d showed much higher level, indicating that the antitumor efficiency
of therapeutic agents was affected by the NIR mediated drug release (Figure 6D).
Specially, the experimental group showed an amazing performance of tumor ablation
and the tumor inhibition reached by 81.4% after 24 days of post-injection (Figure 6E,
6F). The results of gross examination and weights of the mice also showed that
FeTB2@DHA-INPs significantly inhibited tumor growth compared to other treatment
groups (***p < 0.001, Figure 6C, 6D).In addition, when compared with group c, the
results in group b (Free FeTB2) showed relatively slighter tumor weight change during
the 24 days of treated period, indicating the prepared nanosystems can effectively
prevent drug molecules from being removed (Figure 6C, 6D).
Next, the H&E staining was carried out to evaluate the ROS mediated antitumor
14
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 28
1
2
3
4
5
6
7
8
9
effect and potential toxicity of FeTB2@DHA-INPs. As shown in Figure 6G, the results
of group e exhibited more effective antitumor effects and clearly showed tumor necrosis,
and destroy of blood vessels. The toxic side effects of prepared nanoparticles should
not be negligible, however, the toxicity of FeTB2@DHA-INPs in the major organs in
all experimental groups showed no significant organ damage or inflammation lesion,
which attributed to ROS mediated oxidative damage. These satisfactory results fully
demonstrated the excellent biocompatibility of FeTB2@DHA-INPs and showed great
potential as a nanotheranostic agent for ROS mediated anticancer therapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4. Conclusion
In summary, we successfully developed a multifunctional FeTB2-engineered and
DHA-grafted nanodrug agent, to construct an innovative ROS-killing nano-delivery
system (FeTB2@DHA-INPs). The encapsulated ICG enables the nanosystems to have
NIR-responsive drug release. The released FeTB2 could not only directly kill tumor
cells by inducing DNA arrest, but the Fe3+ catalyzes the Fenton reaction to increase
ROS levels, which further leads to oxidative damage. Moreover, the surface-loaded
DHA further complements the intracellular H2O2 and effectively enhances ROS yields.
When compared with free FeTB2, FeTB2@DHA-INPs exhibited much lower
cytotoxic in normal cells and superior antitumor activity at the same dose of FeTB2.
Moreover, the polymer chains design enables the nanosystem prolonged circulation in
the blood and effective accumulation in tumor cells. These results provide an
alternative strategy to antitumor with ROS mediated oxidative damage, which showed
great potential in clinical application.
Supporting Information
Synthesis procedures and other supplementary data.
Statement
All animal experiments were complied with the Regulations on the Administration
of Laboratory Animals and the relevant national laws and regulations, and performed
under the guidance approved by the Laboratory Animal Ethics Committee at School of
Medicine, Southeast University (Nanjing, China).
15
ACS Paragon Plus Environment

Page 17 of 28
Molecular Pharmaceutics
1
2
3
4
5
Acknowledgements
6
7
8
9
We are highly grateful to the financial support from the National Natural Science
Foundation of China (Grant Nos. 21905138 and 21628101), the National Natural
Science Foundation of Jiangsu Province (Grant Nos. BK20190756), the project funded
by China Postdoctoral Science Foundation (No. 2019M651841), the National Key
Research and Development Program of China (2016YFD0600801), the Top-notch
Academic Programs Project of Jiangsu Higher Education Institutions (TAPP, Grant
Nos. PPZY2015C221) and Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
References
(1) Dolmans, D.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003,
3, 380-387.
(2) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Golab, J. Photodynamic
Therapy of Cancer: An Update. Ca-Cancer J. Clin. 2011, 61, 250-281.
(3) Kim, S. U.; Park, Y. H.; Min, J. S.; Sun, H. N.; Han, Y. H.; Hua, J. M.; Lee, T. H.; Lee, S. R.;
Chang, K. T.; Kang, S. W.; Kim, J. M.; Yu, D. Y.; Lee S. H.; Lee, D. S. Peroxiredoxin I is A
ROS/p38 MAPK-Dependent Inducible Antioxidant that Regulates NF-κB-Mediated iNOS
Induction and Microglial Activation J. Neuroimmunol 2013, 259, 26-36.
(4) Park, Y. H.; Kim, S. U.; Lee, B. K.; Kim, H. S.; Song, I. S.; Shin, H. J.; Han, Y. H.; Chang, K. T.;
Kim, J. M.; Lee, D. S.; Kim, Y. H.; Choi, C. M.; Kim, B. Y.; Yu, D. Y. Prx I Suppresses K-ras-
Driven Lung Tumorigenesis by Opposing Redox-Sensitive ERK/Cyclin D1 Pathway. Antioxid.
Redox Sig. 2013, 19, 482-496.
(5) Murphy, M. P.; Holmgren, A.; Larsson, N. G.; Halliwell, B.; Chang, C. J.; Kalyanaraman, B.; Rhee,
S. G.; Thornalley, P. J.; Partridge, L.; Gems, D.; Nystrom, T.; Belousov, V.; Schumacker, P. T.;
Winterbour, C. C. Unraveling the Biological Roles of Reactive Oxygen Species. Cell Metab. 2011,
13, 361-366.
(6) Dixon, S. J.; Lemberg, K. M.; Lamprecht, M. R.; Skouta, R.; Zaitsev, E. M.; Gleason, C. E.; Patel,
D. N.; Bauer, A. J.; Cantley, A. M.; Yang, W. S.; Morrison III, B.; Stockwell, B. R. Ferroptosis:
An Iron-Dependent Form of Nonapoptotic Cell Death. Cell 2012, 149, 1060-1072.
16
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 28
1
2
3
4
5
6
7
8
(7) Shi, L. L.; Guan, Q. H.; Gao, X. H.; Jin, X.; Xu, L.; Shen, J.; Wu, C. W.; Zhu, X. Y.; Zhang, C.
Reaction-based color-convertible fluorescent probe for ferroptosis identification. Anal. Chem. 2018,
90, 9218−9225.
9
(8) Zhu, T.; Shi, L. L.; Yu, C. Y.; Dong, Y. B.; Qiu, F.; Shen, L. Y.; Qian, Q. H.; Zhou, G. Y.; Zhu, X.
Y. Ferroptosis promotes photodynamic therapy: supramolecular photosensitizer-inducer nanodrug
for enhanced cancer treatment. Theranostics 2019, 9, 3293-3307.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(9) Zhou, Z.; Song, J.; Tian, R.; Yang, Z.; Yu, G. C.; Lin, L. S.; Zhang, G. F.; Fan, W. P.; Zhang, F.
W.; Niu, G.; Nie, L. M.; Chen, X. Y. Activatable Singlet Oxygen Generation from Lipid
Hydroperoxide Nanoparticles for Cancer Therapy. Angew. Chem. Int. Edit. 2017, 129, 6492-6496.
(10) Hu, P.; Wu, T.; Fan, W. P.; Chen, L.; Liu, Y. Y.; Ni, D. L.; Bu, W. B.; Shi, J. L. Near Infrared-
assisted Fenton Reaction for Tumor-Specific and Mitochondrial DNA-targeted Photochemotherapy.
Biomaterials 2017, 141, 86-95.
(11) Tang, Z. M.; Zhang, H. L.; Liu, Y. Y.; Ni, D. L.; Zhang, H.; Zhang, J. W.; Yao, Z. W.; He, M. Y.;
Shi, J. L.; Bu, W. B. Antiferromagnetic Pyrite as the Tumor Microenvironment-Mediated
Nanoplatform for Self-Enhanced Tumor Imaging and Therapy. Adv. Mater. 2017, 29, 1701683.
(12) Ahmed, K.; Yellamelli, V. V. S.; Mohammed, N. A.; Sultana, F.; Methuku, K. R. Recent Advances
on Structural Modifications of Benzothiazoles and Their Conjugate Systems as Potential
Chemotherapeutics. Expert Opin Investig Drugs 2012, 21, 619-635.
(13) Dubey, R.; Shrivastava, P. K.; Basniwal, P. K.; Bhattacharya, S.; Moorthy, N. S. 2-(4-Aminophenyl)
Benzothiazole: A Potent and Selective Pharmacophore with Novel Mechanistic Action towards
Various Tumour Cell Lines. Mini-Rev Med Chem 2006, 6, 633-637.
(14) Noolvi, M. N.; Patel, H. M.; Kaur, M. Benzothiazoles: Search for Anticancer Agents. Eur J Med
Chem 2012, 54, 447-462.
(15) Sleebs, B. E.; Kersten, W. J.; Kulasegaram, S.; Nikolakopoulos, G.; Hatzis, E.; Moss, R. M.; Parisot,
J. P.; Yang, H.; Czabotar, P. E.; Fairlie, W. D.; Lee, E. F.; Adams, J. M.; Chen, L.; Delft, M. F. V.;
Lowes, K. N.; Wei, A.; Huang, D. C.; Colman, P. M.; Street, I. P.; Baell, J. B.; Watson, K.; Lessene,
G. Discovery of Potent and Selective Benzothiazole Hydrazone Inhibitors of BCL-XL. J Med Chem
2013, 56, 5514-5540.
(16) Lessene, G.; Czabotar, P. E.; Sleebs, B. E.; Zobel, K.; Lowes, K. N.; Adams, J. M.; Baell, J. B.;
Colman, P. M.; Deshayes, K.; Fairbrother, W. J.; Flygare, J. A.; Gibbons, P.; Kersten, W. J.;
17
ACS Paragon Plus Environment

Page 19 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
Kulasegaram, S.; Moss, R. M.; Parisot, J. P.; Smith, B. J.; Street, I. P.; Yang, H.; Huang, D. C.;
Watson, K. G. Structure-Guided Design of A Selective BCL-XL Inhibitor. Nat Chem Biol 2013, 9,
390-397.
9
(17) Racane, L.; Pavelic, S. K.; Nhili, R.; Depauw, S.; Paul-Constant, C.; Ratkaj, I.; David-Cordonnier,
M. H.; Pavelic, K.; Tralic-Kulenovic, V.; Karminski-Zamola, G. New Anticancer Active and
Selective Phenylene-Bisbenzothiazoles: Synthesis, Antiproliferative Evaluation and DNA binding.
Eur J Med Chem 2013, 63, 882-891.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(18) Hussein, B. H.; Azab, H. A.; El-Azab, M. F.; El-Falouji, A. I. A Novel Anti-tumor Agent, Ln (Ⅲ)
2-Thioacetate Benzothiazole Induces Anti-angiogenic Effect and Cell Death in Cancer Cell Lines.
Eur J Med Chem 2012, 51, 99-109.
(19) Singh, N. P.; Lai, H. Selective Toxicity of Dihydroartemisinin and Holotransferrin toward Human
Breast Cancer Cells. Life Sciences 2001, 70, 49-56.
(20) Kang, M. H.; Reynolds, C. P. Bcl-2 Inhibitors: Targeting Mitochondrial Apoptotic Pathways in
Cancer Therapy. Clin Cancer Res 2009, 15, 1126-1132.
(21) Nariculam, J.; Freeman, A.; Bott, S.; Munson, P.; Cable, N.; Brookman-Amissah, N.; Williamson,
M.; Kirby, R. S.; Masters, J.; Feneley, M. Utility of Tissue Microarrays for Profiing Prognostic
Biomarkers in Clinically Localized Prostate Cancer: the Expression of BCL-2, E-cadherin, Ki-67
and p53 as Predictors of Biochemical Failure after Radical Prostatectomy with Nested Control for
Clinical and Pathological Risk Factors. Asian J Androl 2009, 11, 109-118.
(22) Zhang, X. Y.; Guo, Z. Q.; Ji, S. Q.; Zhang, M.; Jiang, N.; Li, X. S.; Zhou, L. Q. Small Interfering
RNA Targeting HMGN5 Induces Apoptosis via Modulation of A Mitochondrial Pathway and BCL-
2 Family Proteins in Prostate Cancer Cells. Asian J Androl 2012, 14, 487-492.
(23) Hussein, B. H.M.; Azab, H. A.; El-Azab, M. F.; El-Falouji, A. I. A novel anti-tumor agent, Ln(III)
2-thioacetate benzothiazole induces anti-angiogenic effect and cell death in cancer cell lines. Eur J
Med Chem 2012, 51, 99-109.
(24) Gao, Z. G., Li, Y. J.; You, C. Q.; Sun, K.; An, P. J.; Sun, C.; Wang, M. X.; Zhu, X. L.; Sun, B. W.
Iron Oxide Nanocarrier-Mediated Combination Therapy of Cisplatin and Artemisinin for
Combating Drug Resistance Through Highly Increased Toxic Reactive Oxygen Species Generation.
ACS Appl. Bio Mater. 2018, 1, 270-280.
(25) You, C. Q.; Wu, H. S.; Wang, M. X.; Zhang, Y. W.; Wang, J. W.; Luo, Y. H.; Zhai, L. H.; Sun, B.
W.; Zhang, X. Y.; Zhu, J. Near-Infrared Light and pH Dual-Responsive Targeted Drug Carrier
18
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 28
1
2
3
4
5
6
Based on Core-CrosslinkedPolyaniline Nanoparticles for Intracellular Delivery of Cisplatin. Chem.
Eur. J. 2017, 23, 5352-5360.
7
8
9
(26) Tang, G. S.; Xiong, R. H.; Lv, D.; Xu, R. X.; Braeckmans, K.; Huang, C. B.; De Smedt, S. C. Gas-
Shearing Fabrication of Multicompartmental Microspheres: A One-Step and Oil-Free Approach.
Advanced Science 2019, 6, 1802342-1802351.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Hua, D. W.; Liu, Z. C.; Wang, F.; Gao, B. H.; Chen, F.; Zhang, Q. L.; Xiong, R. H.; Han, J. Q.;
Samal, S. K.; De Smedt, S. C.; Huang, C. B. PH Responsive Polyurethane (core) and Cellulose
Acetate Phthalate(shell) Electrospun Fibers for Intravaginal Drug Delivery. Carbohydrate
Polymers 2016, 151, 1240–1244
(28) You, C. Q.; Gao, Z. G.; Wu, H. S.; Sun, K.; Ning, L. K.; Lin, F.; Sun, B. W.; Wang, F. Reactive
Oxygen Species Mediated Theranostics Using A Fenton Reaction Activable Lipopolymersome. J.
Mater. Chem. B 2019, 7, 314-323.
(29) Jing, X. N.; Zhi, Z.; Jin, L. M.; Wang, F.; Wu, Y. S.; Wang, D. Q.; Yan, K.; Shao, Y. Q.; Meng, L.
J. PH/Redox Dual-Stimuli-Responsive Cross-linked Polyphosphazene Nanoparticles for
Multimodal Imaging-Guided Chemo-Photodynamic Therapy. Nanoscale 2019, 11, 9457-9467.
(30) Gao, S. T.; Tang, G. S.; Hua, D. W.; Xiong, R. H.; Han, J. Q.; Jiang, S. H.; Zhang, Q. L.; Huang,
C. B. Stimuli-responsive bio-based polymeric systems and their applications. Journal of Materials
Chemistry B 2019, 7, 709–729.
(31) You, C. Q.; Wu, H. S.; Gao, Z. G.; Sun, K.; Chen, F. H.; Tao, W. A.; Sun, B. W. Subcellular Co-
delivery of Two Different Site-Oriented Payloads Based on Multistage Targeted Polymeric
Nanoparticles for Enhanced Cancer Therapy. J. Mater. Chem. B 2018, 6, 6752-6766.
(32) You, C. Q.; Wang, M. X.; Wu, H. S.; An, P. J.; Pan, M. M.; Luo, Y. H.; Sun, B. W. Near Infrared
Radiated Stimulus-Responsive Liposomes Based on Photothermal Conversion as Drug Carriers for
Co-delivery of CJM126 and Cisplatin. Materials Science and Engineering C 2017, 80, 362-370.
(33) Liu, Z. J.; Le, Z. C.; Lu, L. J.; Zhu, Y.; Yang, C. B.; Zhao, P. F.; Wang, Z. Y.; Shen, J.; Liu, L. X.;
Chen, Y. M. Scalable Fabrication of Metal-Phenolic Nanoparticles by Coordination-Driven Flash
Nanocomplexation for Cancer Theranostics. Nanoscale 2019, 11, 9410-9421.
(34) You, C. Q.; Yu, J.; Sun, Y.; Luo, Y. H.; Zhang, X. Y.; Zhu, J.; Sun, B. W. Enhanced Cytotoxicity
by A Benzothiazole-containing Cisplatin Derivative in Breast Cancer Cells. New J. Chem. 2017,
41, 773-785.
19
ACS Paragon Plus Environment

Page 21 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1 Self-assembly of ICG and FeTB2-embedded DHA-grafted nanoparticles (FeTB2@DHA-
INPs).
20
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1 A, Size distribution of FeTB2@INPs and FeTB2@DHA-INPs determined by DLS. B,
UV−vis −NIR spectra of FeTB2 in methanol, Lecithin, DHA, DSPE-PEG2000 and FeTB2@DHA-
INPs suspensions in phosphate buffer (pH = 7.4). C, TEM image of FeTB2@DHA-INPs. Scale bar:
100 nm. D, TEM image of FeTB2@DHA-INPs in PBS (pH = 7.4) after 5 min post-irradiation. Scale
bar: 200 nm. E, The Zeta potential on the surface of FeTB2@INPs and FeTB2@DHA-INPs. F,
Images of nanoparticles suspension at 25 °C (pH = 7.4): a, DHA-INPs; b, FeTB2@DHA-INPs; c,
FeTB2@DHA-INPs+NIR.
21
ACS Paragon Plus Environment

Page 23 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2 A, The NIR induced temperature curves of nanoparticles suspension with different
concentrations (10, 20, 40 μg mL-1). B, The pictures of FeTB2@DHA-INPs suspension at different
time points (0, 1, 2, 3, 4, 5 min) imaged by infrared (IR) thermal camera. C, NIR dependent release
of FeTB2 from the FeTB2@DHA-INPs at different pH (7.4, 6.8, 5.5). D, TEM image of
FeTB2@DHA-INPs in PBS (pH=7.4) after 48 h post-irradiation.
22
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3 A, CLSM images of A549 and MCF-7 cells incubated with FeTB2@DHA-INPs for
different period of time at an equivalent ICG concentration of 50 μg mL−1 (Scale bar, 20 μm). Flow
cytometric analysis (B) and quantitation (C) of the ICG fluorescence intensity in A549 and MCF-7
cells (A549: a, 0.5 h, c, 3.0 h; MCF-7: b, 0.5 h, d, 3.0 h). D, Internalization of FeTB2@DHA-INPs
suspension (equivalent to 50 μg mL−1 ICG) by MCF-7 cells for 3 h, analyzed by CLSM (Scale bar,
40 μm).
23
ACS Paragon Plus Environment

Page 25 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4 Evaluation the capability of ROS generation of FeTB2@DHA-INPs in vitro. A, CLSM
images of MCF-7 cells incubated with FeTB2@DHA-INPs for different concentrations (10, 20, 30
μg mL-1) and quantitated analysis (D). B, Flow cytometric analysis of ROS levels in MCF-7 cells
with different incubation time (a, 0.5 h; b, 1.0 h; c, 1.5 h; d, 3.0 h) and quantitated analysis (E). C,
Flow cytometric analysis of ROS levels in MCF-7 cells with different nanoparticle formations (a,
Control group; b, Free FeTB2; c, FeTB2@INPs; d, FeTB2@DHA-INPs; e, FeTB2@DHA-INPs+NIR)
and quantitated analysis (F). (n = 3), *: P < 0.05, **: P < 0.01 and ***: P < 0.001.
24
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5 Quantitative evaluation of cell viability for A549 cells (A) and MCF-7 cells (B) treated
with different nanoparticle formulations for 24 h. C, Quantitative evaluation of cell viability for
RGC-5 cells treated with FeTB2@DHA-INPs. D, Quantitative evaluation of cell viability for MCF-
7 cells treated with NPs for different DHA concentrations. E, The apoptosis of MCF-7 cells treated
with FeTB2@DHA-INPs. (n = 3). F, Effect of FeTB2 and FeTB2@DHA-INPs on cell cycle arrest
in MCF-7 cells and quantitative analysis (G). N.S.: P > 0.05, no significance, *: P < 0.05, **: P <
0.01, ***: P < 0.001.
25
ACS Paragon Plus Environment

Page 27 of 28
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6 A, In vivo fluorescence imaging of MCF-7 breast tumor-bearing nude mice and major
organs of mice sacrificed 24 h after intratumoral injection of FeTB2@DHA-INPs. B, Quantitative
evaluation of fluorescence intensity of tumor and major organs of mice sacrificed 24 h after
intratumoral injection. C, Relative tumor volume growth curves in different treatment groups of
tumor-bearing mice (were normalized to their initial sizes). D, Mice weight growth curves in
different treatment groups of tumor-bearing mice. E, Representative photographs of mice bearing
26
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 28
1
2
3
4
5
6
7
8
9
MCF-7 breast tumors treated with different NPs suspensions: (a), control group; (b), Free FeTB2;
(c), FeTB2@INPs+NIR; (d), FeTB2@DHA-INPs; (e), FeTB2@DHA-INPs+NIR. F, The tumor
weight in different groups of tumor-bearing mice after incubation for 24 days. G, H&E stained
images of tumors and major organs from mice sacrificed after incubation for 24 days. Error bars
represent the standard deviation of six mice per group. Data are presented as the mean ± SD (n = 6,
*p < 0.05, **p < 0.01, ***p < 0.001. vs. control group).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
ACS Paragon Plus Environment