An azobenzene-based heteromeric prodrug for hypoxia-activated chemotherapy by regulating subcellular localization

Article abstract of DOI:10.1039/c8cc03430c

An azobenzene-based heteromeric prodrug (hNDP) was prepared for targeted chemotherapy against hypoxic tumor. hNDP could divert the parent drug from nucleus to cytoplasm with lower toxicity, while the azoreduction of hNDP in hypoxia would activate the drug with a robust anti-tumor effect by initiating the apoptosis-related biochemical cascades.

Full text of DOI:10.1039/c8cc03430c

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An azobenzene-based heteromeric prodrug for hypoxia-activated
chemotherapy by regulating subcellular localization
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
Shiying Li,‡ Xueyan Jiang,‡ Rongrong Zheng, Shengjia Zuo, Linping Zhao, Guiling Fan,
a
c
a
b
Jinghao Fan, Yonghua Liao, Xiyong Yu* and Hong Cheng*
DOI: 10.1039/x0xx00000x
www.rsc.org/
An azobenzene-based heteromeric prodrug (hNDP) was prepared (TOP2) to create a DOX-DNA-TOP2 ternary complex, causing
for targeted chemotherapy against hypoxic tumor. hNDP could the structural failure of DNA and the irreversible cell
5
,6
divert the parent drug from nucleus to cytoplasm with lower damage.
While it was also found that some subtle
toxicity. While the azoreduction of hNDP in hypoxia would alterations to the molecular structure of DOX could
activate the drug with a robust anti-tumor effect by initiating the unexpectedly divert its transport pathway and subcellular
apoptosis-related biochemical cascades.
localization, facilitating the enhancement of the therapeutic
7
,8
index. Even so, the lack of specificity has limited its cellular
uptake and the accurate subcellular localization of DOX
towards tumor cells. Moreover, the off-target therapeutic
protocols of DOX would also wreak havoc on the nuclear DNA
The cellular uptake and subcellular localization of therapeutic
agents could ultimately influence their action and efficacy.
Enormous efforts have been devoted to chemically modify and
embellish such agents with spatiotemporally controlled
distribution manners, while the results are often
9
,10
of normal cells, resulting in undesirable side effects.
In the past few decades, tumor-specific microenvironments
have been widely used in prodrug activation for the targeted
1
,2
counterintuitive. Additionally, although great progress has
been made in this field by constructing nano-drug delivery
systems, the tumor cell specificity of the therapeutics remains
relatively low and the adverse effects on normal tissues are
1
1-13
treatment.
For instance, the aggressive progression,
aberrant neovasculature and high interstitial pressures of solid
tumors would lead to the formation of hypoxic regions, which
could conversely promote tumor invasion, metastasis and
3
dose-limiting. In view of that the action sites of various
1
4
therapeutic agents are in specified subcellular location, a
promising solution could be attempted by regulating the
cellular localization to make them inactivate in normal cells
while reactivate in tumor cells, which would improve the
therapeutic efficiency and minimize the side effects.
angiogenesis. Owing to its central role in tumor metabolism,
tumor hypoxia was considered as the best validated target for
1
5
the development of novel therapeutic agents. Of note,
several bioreductive prodrugs had been in clinical trial for
tumor hypoxia targeted therapy by the enzymatic reduction
1
6
Doxorubincin (DOX), as an important anthracycline
antibiotic, is one of the most effective classes of antineoplastic
drugs in clinical, which possesses excellent activities against a
mechanism. Even so, this kind of bioreductive systems,
especially for nitrobenzene and nitroimidazole related
hypoxia-responsive systems, were always suffered from some
insurmountable shortcomings such as suboptimal redox
4
broad-spectrum of tumors. It is well-known that DOX is
1
7
inclined to accumulate within nuclear compartment,
intercalate into DNA strand and bind to topoisomerase II
chemistry and complicated synthetic procedures. Given the
alternative strategy of azoreduction under hypoxic condition,
azobenzene-based bioreductive system could be promising for
targeted tumor therapy. In recent decades, it had attracted
great attentions for constructing hypoxia-responsive
1
8
fluorescent probes and nano-drug delivery systems.
However, azobenzene-based bioreductive prodrug was rarely
reported for the precision therapy against hypoxic tumor.
In this work, we reported on
a hypoxia-activated
heteromeric prodrug (designated as hNDP) consisting of
nitrogen mustard, DOX and the azobenzene linker for targeted
tumor therapy by regulating the prodrug activation as well as
the subcellular
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Scheme 1 Schematic illustration of hNDP for hypoxic tumor
targeting therapy by regulating the prodrug activation and
subcellular distribution.
Fig. 1 CLSM images of 4T1 cells, B16 cells and fibroblasts after
treatment with A) DOX (5 μM), B-G) hNDP (20 μM) under
normoxic (B, D and F) or hypoxic (C, E and G) condition. H)
Schematic illustrations (H1) and CLSM images of 4T1 cells after
treatment with hNDP (20 μM) in normoxic (H2 and H3) or
oxygen-poor (H4 and H5) condition using a glass cover. I)
Schematic illustrations (I1) and CLSM images of 4T1 cells after
treatment with hNDP (20 μM) under normoxic condition (I2-I4)
and subsequently incubated in hypoxic condition (I5-I7). J)
distribution (Scheme 1). Under normoxic condition, hNDP
exhibited reduced cytotoxicity by diverting its cellular
distribution from nuclear localization to cytoplasm. However,
the azobenzene bond in hNDP could be reduced under hypoxic
condition, resulting in the release of a potent DNA alkylating
agent N,N-bis(2-chloroethyl)-1,4-benzenediamine and a robust CLSM images (J1 and K1), fluorescence intensity profile
anti-tumor drug DOX, as well as the recovery of nuclear analysis (J2 and K2) and 3D CLSM images (J3 and K3) of 4T1
targeting ability of DOX. By regulating the activation and the cells after treatment with hNDP (20 μM) under J) normoxic or
K) hypoxic condition. The nucleus was stained by Hoechst
3342. Scale bar: 20 μm.
subcellular distribution, hNDP could selectively eliminate
hypoxic tumor cells and leave the normal cells unharmed.
The synthetic procedures of hNDP were outlined in Fig. S1
3
while it still preferred to accumulate into the nucleus of
oxygen-starved cells located underneath the glass cover (Fig.
(
ESI
examined by electrospray ionization-mass spectrometry (ESI-
MS) (Fig. S2, ESI ). Subsequently, the intermediate of
†). Briefly, the precursor of compound 2 was purified and
1
H4, H5 and Fig. 1I5-I7). Moreover, similar results were also
†
obtained by the fluorescence profiles analysis and the 3D
fluorescence imaging (Fig. 1J, K), which strongly indicated the
unique abilities of hNDP for hypoxia regulated prodrug
activation as well as the subcellular redirection.
compound 3 was prepared by chemically modifying the
precursor 2 with P-nitrophenyl chloroformate. Finally, the
hypoxia-activated hNDP was achieved by conjugating the
intermediate with DOX and the obtained hNDP was purified
Encouraged by the above results, we subsequently utilized
and characterized by ESI-MS (Fig. S3, ESI†).
To investigate the cellular uptake and intracellular
distributions, hNDP was respectively co-cultured with mouse
breast tumor cells (4T1 cells), mouse melanoma cells (B16
cells) and human fibroblasts for CLSM imaging. As the control,
we firstly verified the pure nuclear localization ability of native
DOX (Fig. 1A). In sharp contrast, hNDP revealed cytoplasmic
staining only after incubated with 4T1 cells (Fig. 1B), B16 cells
(Fig. 1D) or fibroblasts (Fig. 1F) under normoxic condition. This
significant difference might be attributed to the reduced DNA
binding capacity of hNDP after the blocking modification to the
1
group in DOX. More interestingly, most of the
-NH
2
fluorescence was found to transfer back to the nucleus in all of
the three cell lines after treatment with hNDP under hypoxic
condition (Fig. 1C, E and F), implying that the conjugated DOX
could be liberated and activated by the azoreduction of hNDP
in hypoxia. To further evaluate the hypoxia response ability of
hNDP, oxygen starvation condition were carried out by using
glass covers (Fig. 1H1, I1). As expected, hNDP was mainly
Fig. 2 CLSM images of 4T1 cells after treatment with hNDP (20
μM) under A) normoxic or B) hypoxic condition and stained by
Hoechst 33342 and MitoTracker Green. CLSM images of 4T1
cells after treatment with hNDP (20 μM) under C) normoxic or
D) hypoxic condition and stained by Hoechst 33342 and
localized in cytoplasm under normoxia environment (Fig. 1H2, LysoTracker Green. Scale bar: 20 μm.
H3 and Fig. 1I2-I4),
2
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the commercially available mitochondrial/lysosomal stain of cells, Q3: late apoptosis cells. *p < 0.05 and **p < 0.01 were
determined by a Student’s t-test.
Mito/LysoTracker to further locate the subcellular distribution
of hNDP. Consistent with the previous observations, the red
fluorescence of DOX in hNDP was mainly distributed in the
cytoplasm instead of in nuclei under normoxic condition (Fig.
2
A, C). Moreover, it was found that the cytoplasmic DOX
fluorescence did not match very well with MitoTracker Green
Fig. 2A), but overlay well with LysoTracker Green along with
the incubation time (Fig. 2C and Fig. S4, ESI ), which confirmed
(
†
the endocytosis pathway against 4T1 tumor cells and the off-
target property of hNDP to mithchondria. However, after
incubated with hypoxic 4T1 cells, the original nuclear
localization ability of DOX in hNDP was recovered completely
(Fig. 2B, D), which demonstrated a great potential in hypoxia-
activated tumor cell nuclear targeting chemotherapy.
Having verified the hypoxia regulated nucleus targeting
capability of hNDP, we subsequently studied its anti-
proliferation effect on tumor cells by using MTT assay and flow
cytometry. DOX exhibited the high cytotoxicity whether in
normoxic or hypoxic conditions due to its non-selectivity (Fig.
S5, ESI † ). However, a higher cytotoxicity of hNDP was
Fig. 4 A) Western blot analysis of Bax, Bad and Bcl-2 expression
in 4T1 cells after treatment with hNDP (20 μM) under
normoxic or hypoxic condition. B) The corresponding
quantitative analysis of their relative expression. *p < 0.05 and
identified in hypoxic condition than that of in normoxic
condition (Fig. 3A, B). Moreover, hNDP did not exhibit a
significantly higher anti-proliferation ability against hypoxic
cells, which might be ascribed to the slow activation processes
of hNDP under hypoxia condition. Additionally, similar results
were also detected by flow cytometry analysis (Fig. 3C). After
treatment with hNDP in hypoxic condition, the population of
apoptotic
*
*p < 0.01 were determined by a Student’s t-test. C) The
proposed mechanism of hNDP for hypoxia-activated tumor
therapy by caspases-related apoptosis mechanism.
cells was about 61.5% (Fig. 3C4), which was more than 2-fold
higher than 30.3% for the cells treated with hNDP in normoxic
condition (Fig. 3C3). Furthermore, the different cell status was
also observed by Annexin V-FITC/PI staining and real-time
fluorescence image analysis (Fig. 3C5, C6 and Fig. S6, S7, ESI†).
Based on the above observations, it could be concluded that
hNDP possessed an enhanced anti-proliferation ability against
hypoxic tumor cells, which might be ascribed to the hypoxia-
activated nucleus targeting ability of hNDP. Besides, in view of
the hypoxia nature of tumor microenvironments, the unique
ability of hNDP for hypoxia targeted therapy would provide a
promising strategy for traditional chemotherapeutic agents
used in effective tumor precision therapy.
DOX and nitrogen mustard could interact with nuclear DNA
to induce programmed cell death by apoptosis related
6
).
,19,20
pathways (Fig. S8, ESI
†
To confirm this pathway, we
examined the expression levels of apoptotic-related proteins
in 4T1 cells after incubated with hNDP under normoxic or
hypoxic condition. As illustrated in Fig. 4A and B, a significantly
higher Bad and Bax expressions were observed in Hypoxia
group than that of in normoxia group, demonstrating that
Fig. 3 Cell viability of A) 4T1 cells and B) B16 cells after treated there was an up-regulation impact of cell apoptosis after
with hNDP at gradient concentrations under normoxia or treatment with hNDP under hypoxic condition. Furthermore,
hypoxia. C) Flow cytometry analysis (C1-C4) and the the anti-apoptotic-related protein of Bcl-2 was also detected
corresponding fluorescence images (C5-C6) of 4T1 cells after
as the control. As expected, a lower Bcl-2 expression was
treated with hNDP (20 μM) under normoxia (C3) or hypoxia
found in Hypoxia group. Thus, we would like to highlight that
(C4), and 4T1 cells incubated in normoxia (C1) and hypoxia (C2)
were used as the controls. Q1: live cells, Q2: early apoptosis
hNDP could effectively initiate the apoptosis related pathway
under hypoxic condition with an enhanced anti-proliferation
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ability. And the mechanism of hNDP for hypoxia-activated and kidney) (Fig. S11, ESI
ChemComm
†
), suggesting the good
tumor targeting chemotherapy was proposed in Fig. 4C. In biocompatibility of the heteromeric prodrug.
brief, after internalized into the cytoplasm by endocytosis, the
inactivated hNDP prodrug under
In summary, we confirmed that the azobenzene-based
heteromeric prodrug (hNDP), which carried the
chemotherapeutic drugs of DOX and nitrogen mustard, could
achieve the tumor hypoxia-induced drug release and effective
targeted chemotherapy. Under normoxic condition, hNDP
could inhibit the cellular internalized drugs from entering the
cell nucleus, leading to their inactivation and the significantly
lower cytotoxicity. However, azoreduction-induced prodrug
activation under hypoxic condition would recover the nuclear
targeting ability and then initiate the apoptosis related
biochemical cascades. By regulating the subcellular localization
in this way, hNDP exhibited a robust anti-proliferation ability
against hypoxic tumor with little systemic side effects. The
study reported herein for altering the drug molecular
distribution could be potentially applied towards other off-
target therapeutic agents, which would advance the
Fig. 5 A) The relative tumor volume after post-treatment. B) development in the field of tumor precision therapy.
The average tumor weight and C) the representative tumor
image and their corresponding H&E staining images of the
sacrificed tumor tissues at the 12th day. Scale bar: 50 μm. D)
The relative body weight changes of the mice after post-
treatment. P < 0.05 and P < 0.05 was analyzed by the
Student’s t-test compared with that of PBS and DOX.
This work was financially supported by the National Natural
Science Foundation of China (81330007).
#
+
Conflicts of interest
There are no conflicts to declare.
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parent drugs to take effect. Moreover, the cytoplasmic hNDP
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Graphical Table Content
An azobenzene-based heteromeric prodrug (hNDP) was prepared for tumor targeted chemotherapy
by regulating subcellular localization.
This journal is © The Royal Society of Chemistry 20xx
Chem. Commun., 2013, 00, 1-3 | 5
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