Real-time monitoring of etoposide prodrug activated by hydrogen peroxide with improved safety

Article abstract of DOI:10.1039/c9tb02041a

Etoposide is one of the most used first-line chemotherapeutic drugs. However, its application is still limited by its side effects. Herein, we designed a novel H₂O₂ sensitive prodrug 6YT for selectively releasing the anti-cancer drug etoposide in cancer cells. In this paper, etoposide and a hydrogen peroxide (H₂O₂) sensitive aryl borate ester group were linked by a fluorescent coumarin and finally the prodrug 6YT was generated. The fluorescence of coumarin was quenched before the connected aryl borate ester group was cleaved by H₂O₂. However, in the high level H₂O₂ environment of the tumor, the fluorescence could be activated simultaneously with the release of etoposide, and the drug release state of the prodrug was monitored real-time. With the support of 6YT, we obtained direct and visual evidence of etoposide release in a high H₂O₂ environment both in cells and zebrafish. The prodrug 6YT was also verified with comparable activity and improved safety with etoposide both in cells and in a mouse model. As a safe and effective prodrug, 6YT is expected to be one of the promising candidates in chemotherapy against cancer.

Full text of DOI:10.1039/c9tb02041a

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Real-time monitoring of etoposide prodrug
activated by hydrogen peroxide with improved
safety†
Cite this: J. Mater. Chem. B, 2019,
7, 7548
Jiawen Zhu, Jingting Chen, Dongmei Song, Wenda Zhang, Jianpeng Guo,
Guiping Cai, Yuhao Ren, Chengying Wan, Lingyi Kong * and Wenying Yu
*
Etoposide is one of the most used first-line chemotherapeutic drugs. However, its application is still
limited by its side effects. Herein, we designed a novel H2O2 sensitive prodrug 6YT for selectively
releasing the anti-cancer drug etoposide in cancer cells. In this paper, etoposide and a hydrogen
peroxide (H2O2) sensitive aryl borate ester group were linked by a fluorescent coumarin and finally the
prodrug 6YT was generated. The fluorescence of coumarin was quenched before the connected aryl
borate ester group was cleaved by H2O2. However, in the high level H2O2 environment of the tumor,
the fluorescence could be activated simultaneously with the release of etoposide, and the drug release
state of the prodrug was monitored real-time. With the support of 6YT, we obtained direct and visual
evidence of etoposide release in a high H2O2 environment both in cells and zebrafish. The prodrug 6YT
was also verified with comparable activity and improved safety with etoposide both in cells and in a
mouse model. As a safe and effective prodrug, 6YT is expected to be one of the promising candidates in
chemotherapy against cancer.
Received 18th September 2019,
Accepted 25th October 2019
DOI: 10.1039/c9tb02041a
rsc.li/materials-b
designed a novel H2O2-response prodrug to selectively release
etoposide in cancer cells. Abundant studies indicated that
Introduction
Over the past few decades, prodrug design has been developed1,2 tumor cells and their microenvironment are different from
to overcome many problems of clinical drugs, such as high normal cells in many aspects, including lower pH,10,11 over-
toxicity, poor selectivity and uncontrolled release. Etoposide is a expressed enzymes,12–15 a higher level of glutathione (GSH)16,17
semisynthetic derivative of podophyllotoxin,3 and one of the first and reactive oxygen species (ROS).18,19 Generally, ROS including
identified topoisomerase II inhibitors, which was widely used in hydrogen peroxide (H2O2), superoxide anions (O2ꢀ) and hydroxyl
cancer therapy, especially in acute myeloid leukaemia, Hodgkin’s radical (ꢁOH), and H2O2 play a major role due to their chemical
or non-Hodgkin’s lymphoma, lung cancer, gastric cancer, breast stability, wide diffusion in and out of cells and tissues and the fact
cancer and ovarian cancer. Etoposide can cause cell death by that they can be generated from nearly all sources of oxidative
forming a ternary complex with topoisomerase II and DNA.4 stress.20 Therefore, H2O2 plays an important role in ROS depen-
However, obvious side effects like cardiac, hematological and dent prodrugs as the therapeutic target. The aryl boronic acids
gastrointestinal toxicity of etoposide have seriously limited its and their esters can be easily oxidized and cleaved by H2O2.21
use in clinical therapy.5–7 Over the years, several researchers In addition, boronic acids and esters are intrinsically nontoxic,
have tried to overcome this problem. In 2013, Linyong Zhu and and their metabolite, boric acid, is considered to be nontoxic to
coworkers reported a photo-trigger etoposide prodrug.8 And in humans.22 Depending on the above factors, boronic acids and
2017, Ping Shi and coworkers reported another etoposide their esters were widely used in the design of tumor targeted
prodrug that could be released in the presence of GSH.9 prodrugs22–26 and fluorescent probes.27–29 Besides in vitro
However, the in vivo toxicity of their prodrugs was not studied experiments, we also performed antitumor and safety studies
and their activities were mainly verified in vitro. Herein, we in vivo, which further verified the feasibility of our prodrug
design.
There is still a lack of simple and effective means for the
prodrugs to detect drug release and distribution directly. Therefore,
it is of major significance to create a real-time monitoring prodrug
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China. E-mail: cpu_lykong@126.com,
by connecting it with a fluorophore. Herein, we report a novel
ywy@cpu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb02041a prodrug to specifically release the antitumor drug etoposide,
7548 | J. Mater. Chem. B, 2019, 7, 7548--7557
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 1 (A) UV-absorption and (B) fluorescence emission spectra of com-
pound 6YT (10 mM, lex = 320 nm, lem = 458 nm) before and after the
addition of H2O2 (500 mM) in PBS buffer after 60 min (pH 7.4, 0.1% DMSO).
Scheme 1 Structure and mechanism of H2O2 prodrug for etoposide
release.
accompanied by in situ fluorescence release in the presence of
H2O2. In this study, the fluorescence of 6YT was detected both
in living cells and zebrafish, to further verify that the prodrug
6YT could react with H2O2 both in vitro and in vivo.
Spectral properties of 6YT towards H2O2
Firstly, we investigated the spectral properties of compound
6YT. The UV-vis absorption spectra indicate that compound
6YT shows a maximum absorption band at about 320 nm both
before and after the addition of H2O2 (Fig. 1A). Besides, the
fluorescence response was detected and is shown in Fig. 1B,
from which we can see that compound 6YT (10 mM) only shows
a very weak emission at 458 nm, but after addition of H2O2
(500 mM) in PBS buffer, the emission intensity shows a signi-
ficant increment after 60 min.
In addition, we studied the time-dependent fluorescence
spectral change (Fig. 2A and B) of compound 6YT (10 mM) after
addition of H2O2 (500 mM) in PBS buffer. The result showed that
the fluorescence intensity was increased to its maximum at
about 100 min with a 32-fold increment, indicating its potential
for the rapid detection of H2O2 and etoposide release. Moreover,
the fluorescence intensities of 6YT towards H2O2 (0–80 eq.) were
increased in a dose-dependent manner (Fig. 2C and D). As the
H2O2 concentration was gradually increased, the fluorescence
In our study, an aryl borate ester was used as the trigger unit
in response to H2O2, and 7-hydroxy-3-hydroxymethyl-coumarin
(7-OH-COU) was used as the fluorophore unit to monitor the
release of etoposide as well as imaging (Scheme 1). When
substituted with an aryl borate ester group, the fluorescence
of A (6YT) is quenched. However, when the aryl borate ester
group is cleaved by H2O2, the phenol intermediate B is quickly
produced, and then a series of electron transfer processes start
and B releases a free etoposide and a quinone methide C
through a 1,8-elimination mechanism. Subsequently, the quinone
methide C is activated with H2O to form fluorophore D (7-OH-
COU), which shows a significant increase in fluorescence. This
mechanism has been applied in nitrogen mustard,30–32
diazeniumdiolate,33–35 ciprofloxacin36 and SN-3837 prodrug design.
In order to take full advantage of the fluorescence, we further
studied the drug release aided by the fluorescence, both in cells
and zebrafish. In addition, we evaluated the anticancer efficacy
and safety on different cell lines and on a mouse model with
human colon cancer HCT-116 cells, to prove the activity and safety
of the prodrug 6YT as a chemotherapy drug against cancer.
Results and discussion
Synthetic procedures
Synthesis of compound 6YT involves three stages (Scheme S1,
ESI†). Firstly, fluorophore 3 (7-OH-COU) was synthesized in
three steps. 3-Methyl-7-propionate-coumarin was synthesized
using 2,4-dihydroxybenzaldehyde and sodium propionate, the
methyl in the allyl position was brominated, then the allyl
bromide was substituted by sodium acetate. 7-OH-COU was
obtained after acidization with 2 N HCl. Second, the H2O2-
responsive aryl borate ester was connected to the phenolic
group of 7-OH-COU. And in the last step, the anti-tumor drug
etoposide was linked to it after a chlorination reaction on the
hydroxymethyl group. The compound 6YT was characterized by
1H, 13C NMR and HRMS (Fig. S6–S8, ESI†). All the intermediate
compounds were also characterized by 1H NMR and HRMS
(Fig. S1–S5, ESI†).
Fig. 2 (A) Time-dependent fluorescence spectra of compound 6YT
(10 mM) upon addition of H2O2 (500 mM) in PBS buffer (pH 7.4, 0.1%
DMSO). (B) The fluorescence spectra change of 6YT (lex = 320 nm, lem
=
458 nm) depending on time. (C) Fluorescence spectra changes of
compound 6YT (10 mM) after treatment with increasing concentrations
of H2O2 (0–80 eq.) in PBS buffer after 100 min. (D) The linear correlation
between the fluorescence intensity of 6YT and the concentration of H2O2
(0–80 eq.) in PBS buffer after 100 min.
This journal is ©The Royal Society of Chemistry 2019
J. Mater. Chem. B, 2019, 7, 7548--7557 | 7549

View Article Online
Journal of Materials Chemistry B
Paper
intensity increased as well, and the linear correlation between In addition, the time-dependent release curve was highly con-
the fluorescence intensity of 6YT (458 nm) and the concentration sistent with the fluorescence curve shown in Fig. 2B, indicating
of H2O2 in the range of 0–80 eq. was determined with R2 of the synchronization of fluorescence and drug release. As shown
0.9930. From the results of fluorescence spectra, we find that the in Fig. 4, the retention time of compound 6YT was identified
aryl borate ester group was easily released by H2O2 in a time and at 9.20 min, and after addition of H2O2 (10 mM) for 30 min,
dose-dependent manner. Furthermore, we also evaluated the pH the peak of 6YT completely disappeared, and three new
dependent response of 6YT toward H2O2 to investigate its peaks concomitantly appeared at 4.53 min (peak1, verified to
practicability for biological imaging (Fig. S9, ESI†). There was be 7-OH-COU), 9.22 min (peak2, verified to be etoposide) and
no obvious change of fluorescence signal at 458 nm above the 9.67 min (peak3). In contrast to the corresponding peaks after
pH range of 5.0–9.0, indicating the stable structure of the 60 min and 120 min, we found that peak3 reduced gradually
prodrug during acidic to alkaline conditions. And as expected, whereas peak1 and peak2 increased concomitantly, indicating
after addition of H2O2 (300 mM), the emission intensity increase that peak3 was a phenol intermediate, as shown in Fig. 4.
was sensitive to the pH range from 5.0 to 9.0, with a maximum In addition, the peak of quinone methide C (in Scheme 1) was not
intensity at pH 7.4 and 37 1C. The results indicate that 6YT found, while peak1 (7-OH-COU) increased gradually, indicating
responded well to H2O2 under normal physiological conditions. that the quinone methide C was transient and easily converted to
7-OH-COU. The evolution of these peaks in the HPLC pattern
Selective response of 6YT towards H2O2
indicated a two-step mechanism. First, the aryl borate ester group
was cleaved by H2O2 and the phenol intermediate was formed.
Immediately after that, the spontaneous 1,8-elimination reaction
started and led to the release of the anti-tumor drug etoposide
and coumarinyl quinone methide, which will rearrange itself to the
fluorophore 7-OH-COU spontaneously in an aqueous medium as
shown in Scheme 1. The peaks of released 7-OH-COU and
etoposide were verified by both retention time and HR-ESI-MS.
To further investigate the fluorescence response specificity of
6YT towards H2O2, the fluorescence intensity of 6YT (10 mM)
was detected in the PBS buffer stimulated by other species at
regular time intervals (30, 60 and 90 min). As shown by the
results (Fig. 3), the addition of other species only resulted in a
negligible fluorescence response, and only H2O2 could induce a
remarkable fluorescence response, indicating good selectivity
of 6YT towards H2O2 cleavage.
Cytotoxicity assay
Drug release ability of 6YT in the presence of H2O2
In order to evaluate the cytotoxicity activity of 6YT, the anti-
tumor activities of 6YT and etoposide were assessed in cancer
cell lines (A549 and HCT-116) and a normal cell line (MCF-10A),
using MTT assays. As shown in Fig. 5, compared with etoposide,
6YT exerted similar cytotoxicity activity against both A549 and
HCT-116 cancer cells with IC50 values of 8.91 and 4.81 mM, but
has much lower cytotoxicity toward MCF-10A cells (normal
cells, IC50 = 60.66 mM) than etoposide (3.98 mM), whereas for
etoposide, no such selectivity was observed, and the cytotoxicities
toward A549, HCT-116 and MCF-10A cell lines were similar with
IC50 values of 4.53, 2.63 and 3.98 mM (Table 1), respectively,
In order to evaluate the ability of the compound 6YT to release
free anti-tumor drug etoposide and fluorophore 7-OH-COU
upon addition of H2O2, reversed-phase HPLC was performed
at regular intervals for verification, using methanol/water (50%/
50%) for the first 6 minutes and methanol/water (90%/10%) for
the rest as the mobile phase, with a flow rate of 1 ml minꢀ1. The
time-dependent release curve is shown in Fig. S10 (ESI†),
etoposide can be released from the compound 6YT in the
presence of H2O2 as determined from HPLC measurements,
while no drug release was detected without the addition of H2O2.
Fig. 3 Fluorescence response of compound 6YT (10 mM) after incubated with
various reactive analytes (500 mM) at 37 1C in PBS (pH 7.4) for 30, 60 and
90 min. PBS: PBS buffer; TBHP: tert-butyl hydroperoxide; OClꢀ: sodium hypo-
chlorite; TEMPO: 2,2,6,6-tetramethylpiperidinyloxy; H2O2: hydrogen peroxide;
ꢁOH: hydroxyl radical; tBuOꢀ: tert-butoxy radical; ONOOꢀ: peroxynitrite; GSH:
glutathione; H2O2; Cys: cysteine; Hcy: homocysteine; Fe3+: FeCl3; Fe2+: FeCl2.
Fig. 4 HPLC chromatogram of 6YT (100 mM) incubated with H2O2
(10 mM). Peaks in the chromatograms were detected by monitoring the
absorption at 280 nm.
7550 | J. Mater. Chem. B, 2019, 7, 7548--7557
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 6 (A) ROS difference between MCF-10A, HCT-116 and A549 cell lines
detected by flow cytometry. (B) The relative ROS level between MCF-10A,
HCT-116 and A549 cell lines.
exogenous H2O2 (0 mM, 100 mM and 200 mM) in A549 cells using
ImageXpress Micro Confocal analysis. As shown in Fig. S11
(ESI†), the fluorescence intensity increased with increasing
concentration of H2O2 indicating that the prodrug 6YT shows
good response to exogenous H2O2. Next, we investigated the
response of the prodrug 6YT to endogenous H2O2 at different
time intervals (0 h, 2 h, 4 h, 8 h and 12 h) in A549 and HCT-116
cell lines. As shown in Fig. S12 and S13 (ESI†), the fluorescence
signal increased in a good time-dependent manner. Cancer
cells have a higher level of ROS compared with normal cells and
H2O2 is an important component of ROS. To further investigate
the different ROS content between cancer cells and normal
cells, MCF-10A (normal cell line) cells were also incubated with
the prodrug 6YT at different time intervals (0 h, 2 h, 4 h, 8 h and
12 h) (Fig. S14, ESI†). Compared with MCF-10A cells, the
fluorescence signal intensity in A549 and HCT-116 cells was
much stronger (Fig. 7), indicating that the compound 6YT in
cancer cells was more active than that in normal cells, which is
consistent with the results of cytotoxicity studies in cancer and
normal cells. In addition, we also tested the fluorescence
response at different concentrations of 6YT after incubation in
A549 cell lines for 12 h. The results are shown in Fig. S15 (ESI†)
Fig. 5 Cytotoxicity study of etoposide and 6YT in A549 (A), HCT-116 (B)
and MCF-10A (C) cell lines. Values are presented as means ꢂ standard
deviations of three independent observations.
Table 1 Cytotoxicity activity in different cell lines
IC50 (mM)
Compounds
A549
HCT-116
MCF-10A
6YT
8.91 ꢂ 0.89
4.53 ꢂ 0.58
480
4.81 ꢂ 0.63
2.63 ꢂ 0.35
480
60.66 ꢂ 1.70
3.98 ꢂ 0.43
480
Etoposide
7-OH-COU
Compound 4
480
480
480
Cytotoxicity activity in different cell lines were determined by the MTT
assay, IC50 ꢂ SD (mM), all experiments were independently performed at
least three times.
suggesting that 6YT was selectively activated in cancer cells, which
have comparatively higher ROS levels than normal cells.
To further investigate if the etoposide was the main source
of cytotoxicity, we also tested the cytotoxicity of 7-OH-COU and
compound 4 (structure shown in Scheme S1, which was not
connected to etoposide, ESI†), the IC50 of which were both beyond
80 mM in A549, HCT-116 and MCF-10A cell lines (Table 1),
indicating that etoposide was the only source of cytotoxicity.
ROS level evaluation of different cell lines
To verify that the differences in the cytotoxicity of compound
6YT in different cells result from the ROS difference among
different cell lines, we measured the ROS of different cells by
using the ROS sensitive dye, 20,70-dichloro-fluorescein diacetate
(DCFH-DA) and then detected the levels by flow cytometry using
a BD Accuri C6 flow cytometer. The ROS levels of HCT-116 and
A549 were obviously higher than that in the normal cell line
MCF-10A (see Fig. 6), which was consistent with the cytotoxicity
results, indicating that the high-level ROS in tumor cells
triggered the release of etoposide to kill the cells.
Fig. 7 (A) Fluorescence images of (a) MCF-10A, (b) HCT-116 and (c) A549
cell lines incubated with compound 6YT (10 mM) for 12 h. Scale bar = 20 mm.
(B) The relative fluorescence intensities of (a–c) were measured at three regions
in each dish. Error bars represent standard deviation (n = 3).
Fluorescence imaging of 6YT in cells
Further, we applied compound 6YT (10 mM) to assess the
applicability of a prodrug in increasing the concentration of
This journal is ©The Royal Society of Chemistry 2019
J. Mater. Chem. B, 2019, 7, 7548--7557 | 7551

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 8 Subcellular localization of 6YT in A549 cells. Cells were treated
with 10 mM 6YT for 12 h. Propidium iodide (PI) was used to identify the
nucleus. Scale bar: 20 mm.
and indicate that the fluorescence signal was increased in a dose- Fig. 10 Confocal fluorescence images of zebrafish incubated with H2O2
(300 mM) for 0 (a–c), 10 (d–f), 20 (g–i) and 30 min (j–l), and subsequently
treated with 6YT (10 mM) for 30 min. Scale bar: 500 mm.
dependent manner.
Intracellular localization of compound 6YT in cells
In order to study the intracellular localization of compound
6YT, cell imaging experiments were tested in A549 cells utiliz-
ing the ImageXpress Micro Confocal analysis system. Cells were
incubated with 10 mM 6YT for 12 h, and subsequently, 1 mM
propidium iodide (PI) for 30 min. A significant fluorescence
signal could be observed, which overlapped with the fluores-
cence signal of PI (fluorescence marker for the nucleus) (Fig. 8).
The results suggest that 6YT was preferentially activated in the
nucleus. This result could be explained by the characteristics of
etoposide since the target of etoposide, topoisomerase II, is in
the nucleus; the 6YT or its phenol intermediate was brought
into and aggregated in the nucleus, then the fluorophore 7-OH-
COU and anti-tumor drug etoposide were continuously released
into the nucleus.
In addition, in vivo real-time monitoring of fluorescence was
also performed in zebrafish (Fig. 10). The zebrafish were
pretreated with H2O2 and incubated for 0, 10, 20 and 30 min,
then treated with 6YT for another 30 min. A gradually increased
fluorescence response was observed in zebrafish in a time
dependent manner. The results demonstrate that our prodrug
6YT could respond to H2O2 in zebrafish, and could be utilized
for real-time detection of the release of the drug in zebrafish.
In vivo tumor studies
We further evaluated the in vivo effect against HCT-116 xeno-
grafts in the BALB/c nude mice model. Etoposide was used as
a comparison control. Human colon cancer HCT-116 was
implanted into the hind flank of female nude mice and allowed
establishing a sizeable tumor. After the solid tumor developed,
the mice were randomized and divided into four groups con-
sisting of six mice/group. As shown in Fig. 11, 6YT dramatically
inhibited tumors (46.19% at 10 mg kgꢀ1 and 59.12% at
20 mg kgꢀ1), which was comparable to etoposide (63.23%).
Meanwhile, the body weight of the mice was decreased gradu-
ally when treated with etoposide, however, for the 6YT group,
the body weights increased normally, indicating the better
safety of 6YT than etoposide. Then we further compared the
toxicity of etoposide and 6YT, the prodrug of etoposide, in vivo.
In vivo imaging of 6YT in zebrafish
Next, we explored the potential of 6YT for in vivo imaging
(Fig. 9) in zebrafish. Zebrafish have many advantages as a
fluorescence model animal, such as the 87% homology with
the human genome and transparent embryos, which enables
observation of the fluorescence in real time. The zebrafish were
pretreated with H2O2 and incubated for 30 min, then treated
with 6YT for another 30 min, and a significant fluorescence was
observed. By contrast, the blank zebrafish exhibited almost no
fluorescence, and the zebrafish treated with only probe 6YT
exhibited only a weak fluorescence. These results are consistent
with the observations in living cells, which further confirmed
that the probe 6YT could be exploited to be a promising tool for
monitoring drug release in living systems. Images were taken
using the DAPI channel.
In vivo acute and short-term toxicity studies in mice
Finally, in order to evaluate the toxicity of 6YT in vivo, acute
toxicity and short-term toxicity tests were performed in normal
ICR mice. In the acute toxicity test, mice were divided into eight
treatment (intraperitoneal) groups (six mice/group, three male
and three female) from 52.43 mg kgꢀ1 to 250 mg kgꢀ1 at a
multiple ratio of 0.8. These animals were observed for abnor-
mal behavior and mortality for 2 weeks after treatment. The
results show that the anti-tumor drug etoposide has a maximum
tolerated dose (MTD) of 81.92 mg kgꢀ1, but no death was found
after treatment with 6YT even at a concentration of 250 mg kgꢀ1
,
which indicates at least a 3-fold increase in MTD of 6YT than
etoposide. Moreover, in the short-term toxicity test, thirty mice
were divided into three groups (ten mice/group, five male and five
female) randomly. Group I was treated with saline as the control,
group II was treated with etoposide at a concentration of
Fig. 9 Confocal fluorescence images of zebrafish (a–c) incubated with
PBS for 30 min; (d–f) incubated with 6YT (10 mM) for 30 min; (g–i)
pretreated with H2O2 (300 mM) for 30 min, subsequently treated with
6YT (10 mM) for 30 min. Scale bar: 500 mm.
7552 | J. Mater. Chem. B, 2019, 7, 7548--7557
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
the 6YT group and the control group. Both the acute toxicity and
short-term toxicity tests indicated that compound 6YT is more
safe and less toxic than etoposide. Lastly, this prodrug design
strategy might be applicable to more chemotherapy drugs, such
as Taxol.
Conclusions
In summary, we designed a novel prodrug 6YT for selective
release of etoposide in cancer cells by targeting H2O2. The
emission intensity shows a 32-fold increment after stimulation
by H2O2 in vitro. 6YT shows highly selective toxicity to cancer
cells that overexpress H2O2, whereas neither obvious damage
nor fluorescence signal to normal cells has been observed,
which was verified by both cytotoxicity and microscopic con-
focal studies. We also performed in vivo imaging in zebrafish,
indicating the potential of 6YT to be used for monitoring drug
release in living systems. Finally, further in vivo tumor studies
and toxicity tests were performed to verify that 6YT has comparable
in vivo activity, but much better safety and less toxicity, than
etoposide. As a consequence, as a safe and effective prodrug, 6YT
is expected to act as a chemotherapy drug against cancer.
Fig. 11 In vivo effects of 6YT on tumor growth in the xenografted nude
mice. (A) Anatomical nude mice’s tumor tissue untreated or treated with
6YT (10 and 20 mg kgꢀ1) or etoposide (20 mg kgꢀ1). (B) Inhibition
percentage of mice’s tumor tissues upon treatment of 6YT (10 and
20 mg kgꢀ1) or etoposide (20 mg kgꢀ1), ***p o 0.0001. (C) The growing
curves of mice’s tumor volume. (D) The growing curves of body weight.
40 mg kgꢀ1, which is about half-value of the MTD to avoid acute
death, and group III were treated with 40 mg kgꢀ1 of 6YT,
accordingly. Mice were treated every other day by intraperitoneal
injection for 14 days (Fig. 12A). Body weights were recorded every
other day (Fig. 12B), and there were no obvious differences
between the control group and the 6YT group, but the weights
of the mice in the etoposide group decreased significantly. After
the fifth day, mice in the etoposide group died gradually until all
the mice died by the tenth day. However, no deaths occurred in
the 6YT group, and no obvious differences were observed between
Experimental
Materials
All the solvents and reagents were purchased from commercial
suppliers and used without further purification. The reaction
was monitored by analytical thin layer chromatography using
precoated silica gel GF254 plates (Qingdao Haiyang Chemical
Plant, Qingdao, China) and detected under a UV lamp (254 or
365 nm). Column chromatography was performed with silica
gel (90–150 mesh; Qingdao Marine Chemical Inc.). 1H NMR
and 13C NMR spectra were measured using a Bruker spectro-
meter (500 and 600 MHz) at 25 1C and referenced to TMS.
Chemical shifts are reported in ppm (d) using the residual
solvent line as the internal standard. High-resolution electro-
spray ionization mass spectra (HR-ESI-MS) were recorded using
a Mariner ESI-TOF spectrometer. ROS was detected by flow
cytometry (488 nm excitation and 525 nm emission filters)
using a BD Accuri C6 flow cytometer (Becton & Dickinson
Company, Franklin Lakes, NJ, USA). All pH measurements were
conducted with a sartorius basic pH-meter PB-10. Data were
processed using cell quest software (Becton & Dickinson Com-
pany, Franklin Lakes, NJ). Fluorescence spectra were recorded
using a Shimadzu FR-6000 luminescence spectrometer. HPLC
analysis was performed using Agilent 1100 high-performance
liquid chromatography. Zebrafish images were obtained using
an OLYMPUS SZX2-ILLB microscope. Cells imaging was performed
using ImageXpress Micro Confocal analysis and quantification of
fluorescence intensity was done using ImageJ software.
Fig. 12 Short-term toxicity test. Thirty mice were distributed into three
groups (5 female and 5 male in each group): group I, saline control group;
group II, 40 mg kgꢀ1 etoposide; group III, 40 mg kgꢀ1 6YT. (A) Survival
rate curves of mice treated with 6YT or etoposide. Mice were treated
(intraperitoneal) with 40 mg kgꢀ1 6YT or etoposide every other day.
(B) Body weight curves of the various groups.
General procedure for the preparation of target compounds
Synthesis of compound 133. To a solution of 2,4-dihydroxy-
benzaldehyde (2.07 g, 15 mmol) in propionic anhydride (50 ml)
This journal is ©The Royal Society of Chemistry 2019
J. Mater. Chem. B, 2019, 7, 7548--7557 | 7553

View Article Online
Journal of Materials Chemistry B
Paper
was added sodium propionate (4.32 g, 45 mmol), piperidine 2.4 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 5.15 (s, 2H), 4.58 (d, J = 0.5
(0.5 ml) and the reaction mixture was further heated under Hz, 2H), 1.35 (s, 12H); HRMS (ESI) m/z 431.1640 [M + Na]+
reflux for 12 hours. After the reaction was complete as evi- (calcd for 431.1636, C23H2511BNaO6).
denced by thin layer chromatography analysis, ice water was
Synthesis of compound 5. A solution of compound 4 (0.61 g,
poured into the solution and the precipitated viscous solid was 1.5 mmol) and TEA (0.625 ml, 4.5 mmol) in dry dichloro-
diluted with EtOAc, the organic phase was washed with brine, methane was added SOCl2 (0.164 ml, 2.25 mmol) dropwise
dried over Na2SO4, and then removed by evaporation under and stirred in an ice bath for 2 hours. Then the solvent was
reduced pressure. The crude residue was purified by column removed by evaporation under reduced pressure, and the
chromatography (petroleum ether/ethyl acetate, 4 : 1) to obtain residue was purified using silica gel column chromatography
compound 1 as a white powder (2.12 g, 61%). 1H NMR (petroleum ether/ethyl acetate, 4 : 1) to afford compound 5 as a
(600 MHz, DMSO) d 7.89 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.25 yellow powder (0.525 g, 82%). 1H NMR (500 MHz, DMSO) d 8.18
(d, J = 2.1 Hz, 1H), 7.12 (dd, J = 8.4, 2.2 Hz, 1H), 2.63 (q, J = 7.5 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.7 Hz, 1H), 7.47
Hz, 2H), 2.09 (d, J = 1.1 Hz, 3H), 1.14 (t, J = 7.5 Hz, 3H); HRMS (d, J = 8.0 Hz, 2H), 7.10 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 8.6,
(ESI) m/z 255.0626 [M + Na]+ (calcd for 255.0628, C13H12NaO4). 2.4 Hz, 1H), 5.27 (s, 2H), 4.59 (s, 2H), 1.28 (s, 12H); HRMS (ESI)
Synthesis of compound 235. A solution of compound 1 m/z 427.1478 [M + H]+ (calcd for 427.1482, C23H2511BClO5).
(1.16 g, 5 mmol) and NBS (0.98 g, 5.5 mmol) in CCl4 (25 ml)
Synthesis of compound 6YT38. A mixture of compound 5
was added to AIBN (0.04 g, 0.25 mmol). The mixture was then (0.51 g, 1.2 mmol) and sodium iodide (1.8 g, 12 mmol) in 10 ml
refluxed under an argon atmosphere. After 8 hours, the solvent of acetone was stirred for 1 hour at ambient temperature. The
was evaporated, and the residue was washed with water and reaction mixture was concentrated under reduced pressure and
extracted with ethyl acetate and dried over Na2SO4. Then the diluted with water. The suspension was extracted with ethyl
crude product was added into a solution of NaOAc (0.62 g, acetate, and the organic phase was collected and washed with
7.5 mmol) in acetic acid (25 ml) and refluxed under an air 10% sodium thiosulfate, and then dried over Na2SO4. The
atmosphere for 5 hours. After the reaction was complete as residue was redissolved into a mixture of etoposide (0.71 g,
evidenced using analytical thin layer chromatography, the 1.2 mmol) and K2CO3 (0.5 g, 3.6 mmol) in acetonitrile, the
solution was evaporated and the residue was purified by solvent was stirred at room temperature for 6 hours. After the
column chromatography (petroleum ether/ethyl acetate, 4 : 1) reaction was complete as evidenced by TLC chromatography,
to obtain compound 2 as a yellow powder (0.52 g, 36%). the solvent was removed under reduced pressure. The residue
1H NMR (600 MHz, CDCl3) d 7.74 (s, 1H), 7.50 (d, J = 8.5 Hz, was subjected to silica gel column chromatography (DCM/
1H), 7.13 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.4, 2.2 Hz, 1H), 5.05 MeOH, 20 : 1) to afford 6YT as a yellow powder (0.62 g, 76%).
(d, J = 0.7 Hz, 2H), 2.63 (q, J = 7.5 Hz, 2H), 2.15 (s, 3H), 1.28 (t, J = m.p. 169–170 1C; 1H NMR (500 MHz, DMSO) d 7.90 (s, 1H), 7.70
7.5 Hz, 3H); HRMS (ESI) m/z 313.0684 [M + Na]+ (calcd for (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.0 Hz,
313.0683, C15H14NaO6).
2H), 7.08 (dd, J = 5.2, 2.4 Hz, 1H), 7.01 (dd, J = 6.3, 2.2 Hz, 2H),
Synthesis of compound 3. To a solution of compound 2 6.54 (s, 1H), 6.23 (s, 2H), 6.03 (d, J = 1.4 Hz, 2H), 5.26 (s, 2H),
(0.44 g, 1.5 mmol) in THF (10 ml) was added 2 N HCl (10 ml), 5.23 (d, J = 5.2 Hz, 2H), 4.93 (d, J = 3.4 Hz, 1H), 4.72 (d, J =
and then the reaction was left under stirring at room tempera- 5.1 Hz, 1H), 4.70 (d, J = 2.4 Hz, 2H), 4.57 (d, J = 7.7 Hz, 1H), 4.55
ture for 12 hours. The THF was then evaporated under reduced (d, J = 5.5 Hz, 1H), 4.27 (dd, J = 9.2, 3.6 Hz, 2H), 4.08 (dd, J =
pressure and the mixture was diluted with EtOAc, the organic 10.1, 4.9 Hz, 1H), 3.91 (s, 1H), 3.68 (s, 1H), 3.59 (s, 6H), 3.51
phase was washed with brine, dried over Na2SO4, and then (t, J = 10.1 Hz, 1H), 3.26–3.22 (m, 1H), 3.16 (t, J = 9.3 Hz, 1H),
removed by evaporation under reduced pressure. The residue 3.09–3.04 (m, 1H), 2.88 (ddd, J = 13.9, 11.7, 3.4 Hz, 1H), 1.29
was purified by column chromatography (petroleum ether/ethyl (s, 12H), 1.24 (d, J = 5.0 Hz, 3H). 13C NMR (126 MHz, DMSO) d
acetate, 2 : 1) to obtain compound 3 as a yellow powder (0.18 g, 175.07, 161.53, 155.04, 153.59, 152.44, 148.26, 146.75, 140.46,
1
64%). H NMR (600 MHz, DMSO) d 10.44 (s, 1H), 7.85 (s, 1H), 140.14, 136.90, 135.10, 132.78, 129.97, 129.35, 127.50, 127.23,
7.56 (d, J = 8.5 Hz, 1H), 6.78 (dd, J = 8.5, 2.3 Hz, 1H), 6.72 (d, J = 121.69, 113.59, 113.00, 110.41, 110.26, 108.42, 102.01, 101.93,
2.2 Hz, 1H), 5.33 (s, 1H), 4.30 (d, J = 1.1 Hz, 2H); HRMS (ESI) m/z 101.80, 99.07, 84.19, 80.60, 79.66, 74.89, 73.22, 72.24, 70.14,
215.0316 [M + Na]+ (calcd for 215.0315, C10H8NaO4).
69.50, 67.83, 66.25, 56.28, 43.59, 40.62, 40.53, 40.45, 40.36,
Synthesis of compound 429. A mixture of compound 3 40.29, 40.20, 40.12, 40.03, 39.86, 39.69, 39.53, 37.74, 25.44,
(0.38 g, 2 mmol), K2CO3 (0.41 g, 3 mmol), KI (0.02 g, 0.1 mmol) 25.16, 20.80; HRMS (ESI) m/z 1001.3371 [M + Na]+ (calcd for
and 4-(bromomethyl)benzeneboronic acid pinacol ester (0.65 g, 1001.3374, C52H5511BNaO18).
2.2 mmol) in acetonitrile (15 ml) was refluxed for 5 hours. After
General procedure for spectra measurements
the reaction was complete as evidenced using TLC chromato-
graphy, the solvent was removed by evaporation under reduced If there are no special instructions, both absorption and
pressure. The residue was subjected to silica gel column fluorescence spectra were measured in 10 mM PBS buffer (pH
chromatography (petroleum ether/ethyl acetate, 2 : 1) to afford 7.4, 0.1% DMSO) at 37 1C. A 10 mM stock solution of 6YT was
compound 4 as a yellow powder (0.62 g, 76%). 1H NMR prepared in DMSO and the final test solution of 6YT (10 mM)
(600 MHz, CDCl3) d 7.84 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.43 was obtained by mixing 1 ml of stock solution with 999 ml of PBS
(d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.6 Hz, 1H), 6.92 (dd, J = 8.6, buffer.
7554 | J. Mater. Chem. B, 2019, 7, 7548--7557
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
The stock solutions of various physiologically important The cytotoxic activity was expressed as the IC50 values. All experi-
ꢁ
ꢁ
species were prepared from H2O2, TBHP, NaOCl, OH, OtBu, ments were conducted in triplicate and repeated more than
ONOOꢀ, TEMPO, GSH, Cys, Hcy, Fe2+ and Fe3+.
H2O2, tert-butylhydroperoxide (TBHP), and hypochlorite
(NaOCl) were obtained from 30%, 70%, and 10% aqueous
solutions, respectively.
three times.
ROS level evaluation of different cell lines
ROS was detected by flow cytometry (488 nm excitation and
525 nm emission filters) using a BD Accuri C6 flow cytometer
(Becton & Dickinson Company, Franklin Lakes, NJ, USA). Data
were processed using cell quest software (Becton & Dickinson
Company, Franklin Lakes, NJ). The level of intracellular ROS
was measured by using the ROS sensitive dye, 20,70-dichloro-
fluorescein diacetate (DCFH-DA). Briefly, MCF-10A, HCT-116
and A549 cells were seeded in six-well plates at 3 ꢃ 105 cells per
well, culturing for 20 h, and then washed three times and
incubated with a final concentration of 10 mM DCFH-DA for
30 min at 37 1C in the dark. After incubation, the cells were
washed three times and harvested in free-serum medium. The
fluorescence of 20,70-dichlorofluorescein (DCF) was detected
by flow cytometry (488 nm excitation and 525 nm emission
filters) using a BD Accuri C6 flow cytometer (Becton & Dickinson
Company, Franklin Lakes, NJ, USA). Data were processed using
cell quest software (Becton & Dickinson Company, Franklin Lakes,
NJ). The data represented is an average of 3 repeats and statistical
analysis was done using t-test. ***p o 0.0001.
Hydroxyl radical (ꢁOH), and tert-butoxy radical (ꢁOtBu) were
generated by reaction of 2.5 mM Fe2+ with 500 mM H2O2 or
TBHP, respectively.39
Peroxynitrite solution (ONOOꢀ) was freshly prepared before
use as reported.40
TEMPO stock solutions were prepared by adding 7.8 mg
TEMPO to 100 ml H2O.
GSH, Cys and Hcy stock solutions were prepared by adding
30.6 mg GSH, 6.1 mg Cys and 6.8 mg Hcy to 100 ml H2O,
respectively.
Fe2+ and Fe3+ solutions were prepared by adding 6.3 mg
FeCl2 and 8.1 mg FeCl3 to 100 ml H2O, respectively.
UV-vis absorption spectra were recorded on a Shimadzu
UV-2450 spectrometer. Fluorescence spectra were recorded
using a Shimadzu FR-6000 luminescence spectrometer. The
UV-vis spectra were recorded from 300 to 450 nm, and the
fluorescence spectra were recorded at an emission wavelength
range from 400 to 600 nm and an excitation wavelength of
320 nm (lem = 458 nm; slit widths, 10 nm/10 nm).
Fluorescence image of compound 6YT activated by different
concentrations of exogenous H2O2
Monitoring of etoposide release by RP-HPLC
The release of the anti-tumor drug etoposide from compound
6YT was monitored by reversed-phase HPLC measurement at
regular time intervals using methanol/water (50%/50%) for the
first 6 minutes and methanol/water (90%/10%) for the rest as
the mobile phase, with a flow rate of 1 ml minꢀ1. No drug
release can be detected in the absence of H2O2. HPLC analysis
was performed using Agilent 1100 high-performance liquid
chromatography.
The cells were seeded in 96-well plates in 100 ml of RPMI/DMEM
medium containing 10% FBS at a density of 15 000 cells per
well. The cells were incubated at 37 1C overnight in a humidi-
fied 5% CO2 incubator to attach overnight. After medium
removal, 6YT (10 mM) was added in 200 ml fresh medium
and incubated at 37 1C for 2 h. After that, different concentra-
tions of H2O2 were added (0, 100 and 200 mM) for another 2 h.
Cell imaging experiments were tested utilizing the ImageXpress
Micro Confocal analysis system at Ex/Em of 360 nm/460 nm
and propidium iodide at Ex/Em of 550 nm/620 nm. Quantifica-
tion of fluorescence intensity was done using ImageJ software.
Cell line and cell culture
All the cell lines were purchased from the Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences (Shanghai, China). Both A549 and MCF-10A cell lines were
incubated in Roswell Park Memorial Institute (RPMI) 1640 medium
and HCT-116 cell lines were incubated in Dulbecco’s modified
Eagle’s medium (DMEM), all the media were supplemented with
10% fetal bovine serum (FBS) at 37 1C in a humidified 5% CO2
incubator.
Fluorescence image of compound 6YT activated by endogenous
H2O2 in different cell lines
The cells were seeded in 96-well plates at a density of
15 000 cells per well. The cells were incubated at 37 1C over-
night in a humidified 5% CO2 incubator to attach overnight.
After medium removal, 6YT (10 mM) were added in 200 ml fresh
medium and incubated at 37 1C for different time intervals
(0, 2, 4, 8, 12 h). Cell imaging experiments were tested utilizing
the ImageXpress Micro Confocal analysis system. Quantification
of fluorescence intensity was done using ImageJ software.
Cytotoxicity assay
Cell viability was determined using an MTT colorimetric assay.
The cells were seeded in 96-well plates at a density of 5000 cells
per well. The cells were incubated at 37 1C overnight in a
humidified 5% CO2 incubator to attach overnight. After medium
removal, different concentrations of test compounds were added
Intracellular localization of compound 6YT
in triplicate to the plates in 200 ml fresh medium and incubated Cell imaging experiments were tested in A549 cells utilizing the
at 37 1C for 48 h. MTT was added to evaluate cell viability. The ImageXpress Micro Confocal analysis system. The cells were
absorbance was measured at 570 nm using a microplate reader incubated with 10 mM 6YT for 12 h, and subsequently, 1 mM
(Spectramax Plus 384, Molecular Devices, Sunnyvale, CA, USA). propidium iodide (PI) for 30 min. A significant fluorescence
This journal is ©The Royal Society of Chemistry 2019
J. Mater. Chem. B, 2019, 7, 7548--7557 | 7555

View Article Online
Journal of Materials Chemistry B
Paper
signal could be observed, which was overlapping with the to avoid acute death. Mice were treated every other day by
fluorescence signal of PI (fluorescence marker for nucleus).
intraperitoneal injection for 14 days. The body weights were
recorded every other day.
In vivo imaging of 6YT in zebrafish
Statistical analysis
Wild type zebrafish were obtained from the Nanjing Xinjia
Pharmaceutical Technology Co., Ltd. Zebrafish were fed in E3
embryo media at 28 1C. The 3-day-old zebrafish were incubated
with PBS for 30 min, and then imaged as the blank control
group. Zebrafish were treated with 6YT (10 mM) for 30 min, and
then imaged, as the negative control group. Zebrafish were
pretreated with H2O2 (300 mM) for 30 min, subsequently treated
with 6YT (10 mM) for 30 min, and then imaged, as the experi-
mental group. Before imaging, 1% agarose gel was adopted for
immobilization of the zebrafish. Fluorescence images were
recorded using an OLYMPUS SZX2-ILLB microscope. Images
were taken using the DAPI channel. Scale bar: 500 mm.
GraphPad Prism version 5.0 (GraphPad Software, San Diego,
CA, USA) was used for statistical analysis. Experimental data are
shown as the mean ꢂ standard deviation. p o 0.05 was
considered to indicate a significant difference.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
In vivo tumor studies of 6YT
We are sincerely grateful to Nanjing Xinjia Pharmaceutical
Technology Co., Ltd, for the help with the zebrafish imaging
experiments. This research work was supported by the National
Natural Science Foundation of China (Grant No. 81973180 and
No. 81673298), the Natural Science Foundation of the Jiangsu
Higher Education Institutions of China for Excellent Young
Talents (Grant No. BK20180077), the Program for Changjiang
Scholars and Innovative Research Team in University (IRT_15R63)
and the Drug Innovation Major Project (2018ZX09735002-003).
All of the animal experiments were approved by the Animal
Ethical Committee of China Pharmaceutical University, and the
procedures were performed strictly in accordance with the
guidelines of the National Institutes of Health on the use of
experimental animals (China).
In vivo tumor studies. Six-week-old female nude mice, of
Specified Pathogens Free (SPF) grade, were purchased from the
Model Animal Research Center of Nanjing University (Nanjing,
China). Nude mice were injected in the right flank area with
3 ꢃ 106 human colon cancer HCT-116 cells in 200 ml of PBS.
After 5–10 days, tumors with a diameter of 3 mm were estab-
lished. The mice were divided randomly into three groups
(6 mice/group) and intraperitoneally administered with 6YT
(10 or 20 mg kgꢀ1), etoposide (20 mg kgꢀ1) or vehicle and
monitored every other day. Tumor growth was determined by
tumor volume, which was calculated according to formula
V = 0.52 ꢃ a2 ꢃ b (a is the smallest superficial diameter and
b is the largest superficial diameter). After 19 days of treatment,
tumors were harvested from the killed mice. Tumor weights
were recorded, the data represented is an average of 6 repeats
and statistical analysis was done using t-test. ***p o 0.0001.
Notes and references
1 T. M. Allen and P. R. Cullis, Science, 2004, 303, 1818–1822.
2 M. C. Garnett, Adv. Drug Delivery, 2001, 53, 171–216.
3 X. Yu, Z. Che and H. Xu, Chemistry, 2017, 23, 4467–4526.
4 K. R. Hande, Eur. J. Cancer, 1998, 34, 1514–1521.
5 C. L. Bennett, J. A. Sinkule, R. L. Schilsky, E. Senekjian and
K. E. Choi, Cancer Res., 1987, 47, 1952–1956.
6 S. Sengupta, P. Tyagi, S. Chandra, V. Ko-chupillai and
S. K. Gupta, Pharmacology, 2001, 62, 163–171.
7 J. A. Sinkule, Pharmacotherapy, 1984, 4, 61–73.
8 Q. Lin, C. Bao, Y. Yang, Q. Liang, D. Zhang, S. Cheng and
L. Zhu, Adv. Mater., 2013, 25, 1981–1986.
Acute toxicity test
In the acute toxicity test, six-week-old normal ICR mice, of
Specified Pathogens Free (SPF) grade, were purchased from
Qinglongshan Animal Breeding Ground (Nanjing, China). The
9 Y. Liu, S. Zhu, K. Gu, Z. Guo, X. Huang, M. Wang, H. M.
Amin, W. Zhu and P. Shi, ACS Appl. Mater. Interfaces, 2017,
9, 29496–29504.
mice were divided into eight treatment (intraperitoneal) groups 10 S. Y. Li, L. H. Liu, H. Z. Jia, W. X. Qiu, L. Rong, H. Cheng and
(six mice/group, three male and three female): 52.43, 65.54, X. Z. Zhang, Chem. Commun., 2014, 50, 11852–11855.
81.92, 102.4, 128, 160, 200 and 250 mg kgꢀ1 at a multiple ratio 11 J. Z. Du, X. J. Du, C. Q. Mao and J. Wang, J. Am. Chem. Soc.,
of 0.8. These animals were observed for abnormal behavior and
mortality for 2 weeks after treatment.
2011, 133, 17560–17563.
12 B. Yu, Y. Zheng, Z. Yuan, S. Li, H. Zhu, L. K. De La Cruz,
J. Zhang, K. Ji, S. Wang and B. Wang, J. Am. Chem. Soc.,
2018, 140, 30–33.
Short-term toxicity test
Moreover, in the short-term toxicity test, thirty mice were divided 13 Y. Wu, S. Huang, F. Zeng, J. Wang, C. Yu, J. Huang, H. Xie
into three groups (ten mice/group, five male and five female) and S. Wu, Chem. Commun., 2015, 51, 12791–12794.
randomly. Group I was treated with saline as the control, group II 14 C. E. Callmann, C. V. Barback, M. P. Thompson, D. J. Hall,
and III were treated with etoposide and 6YT at a concentration of
R. F. Mattrey and N. C. Gianneschi, Adv. Mater., 2015, 27,
4611–4615.
40 mg kgꢀ1, which was about half-value of the MTD of etoposide,
7556 | J. Mater. Chem. B, 2019, 7, 7548--7557
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Paper
Journal of Materials Chemistry B
15 X. Zhang, X. Li, Z. Li, X. Wu, Y. Wu and Q. You, Org. Lett., 28 G. C. Van de Bittner, E. A. Dubikovskaya, C. R. Bertozzi and C. J.
2018, 20, 3635–3638. Chang, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 21316–21321.
16 H. Ma, J. Zhang, Z. Zhang, Y. Liu and J. Fang, Chem. 29 L. Yuan, W. Lin, Y. Xie, B. Chen and S. Zhu, J. Am. Chem.
Commun., 2016, 52, 12060–12063. Soc., 2012, 134, 1305–1315.
17 B. Zhang, C. Ge, J. Yao, Y. Liu, H. Xie and J. Fang, J. Am. 30 R. Weinstain, E. Segal, R. Satchi-Fainaro and D. Shabat,
Chem. Soc., 2015, 137, 757–769. Chem. Commun., 2010, 46, 553–555.
18 Y. Kuang, K. Balakrishnan, V. Gandhi and X. Peng, J. Am. 31 B. Li, P. Liu, H. Wu, X. Xie, Z. Chen, F. Zeng and S. Wu,
Chem. Soc., 2011, 133, 19278–19281. Biomaterials, 2017, 138, 57–68.
19 A. M. Durantini, L. E. Greene, R. Lincoln, S. R. Martinez and 32 M. Ni, W. J. Zeng, X. Xie, Z. L. Chen, H. Wu, C. M. Yu and
G. Cosa, J. Am. Chem. Soc., 2016, 138, 1215–1225. B. W. Li, Chin. Chem. Lett., 2017, 28, 1345–1351.
20 Y. J. Lee and E. Shacter, Free Radical Biol. Med., 2000, 29, 33 K. K. Behara, Y. Rajesh, Y. Venkatesh, B. R. Pinninti, M. Mandal
684–692. and N. D. P. Singh, Chem. Commun., 2017, 53, 9470–9473.
21 A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. 34 G. Ravikumar, M. Bagheri, D. K. Saini and H. Chakrapani,
Chem. Res., 2011, 44, 793–804. Chem. Commun., 2017, 53, 13352–13355.
22 Y. Liao, L. Xu, S. Ou, H. Edwards, D. Luedtke, Y. Ge and 35 G. Ravikumar, M. Bagheri, D. K. Saini and H. Chakrapani,
Z. Qin, ACS Med. Chem. Lett., 2018, 9, 635–640. ChemBioChem, 2017, 18, 1529–1534.
23 M. Li, S. Li, H. Chen, R. Hu, L. Liu, F. Lv and S. Wang, ACS 36 K. A. Pardeshi, T. A. Kumar, G. Ravikumar, M. Shukla,
Appl. Mater. Interfaces, 2016, 8, 42–46.
G. Kaul, S. Chopra and H. Chakrapani, Bioconjugate Chem.,
2019, 30, 751–759.
37 E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K. S.
Hong and J. S. Kim, J. Am. Chem. Soc., 2014, 136, 13888–13894.
24 H. Hagen, P. Marzenell, E. Jentzsch, F. Wenz, M. R. Veldwijk
and A. Mokhir, J. Med. Chem., 2012, 55, 924–934.
25 J. Peiro Cadahia, J. Bondebjerg, C. A. Hansen, V. Previtali,
A. E. Hansen, T. L. Andresen and M. H. Clausen, J. Med. 38 L. Li, Z. Li, W. Shi, X. Li and H. Ma, Anal. Chem., 2014, 86,
Chem., 2018, 61, 3503–3515. 6115–6120.
26 L. Wang, S. Xie, L. Ma, Y. Chen and W. Lu, Eur. J. Med. 39 C. Chung, D. Srikun, C. S. Lim, C. J. Chang and B. R. Cho,
Chem., 2016, 116, 84–89. Chem. Commun., 2011, 47, 9618–9620.
27 L. Yi, L. Wei, R. Wang, C. Zhang, J. Zhang, T. Tan and Z. Xi, 40 H. Zhu, Z. Zhang, S. Long, J. Du, J. Fan and X. Peng, Nat.
Chemistry, 2015, 21, 15167–15172.
Protoc., 2018, 13, 2348–2361.
This journal is ©The Royal Society of Chemistry 2019
J. Mater. Chem. B, 2019, 7, 7548--7557 | 7557