Development of hypoxia-triggered prodrug micelles as doxorubicin carriers for tumor therapy

Article abstract of DOI:10.1039/c4ra14875d

Hypoxia has a major role in tumor development and resistance to therapy. Therefore, the effective targeting and killing of hypoxic tumor cells is a key to successful tumor control. Here, we report the hypoxia-responsive prodrug micelles to deliver hydrophobic anticancer drug, which can selectively release the drugs to treat hypoxic tumor cells in a combined way. For this purpose, an azobenzene (AZO) bond, which imparts hypoxia sensitivity and specificity as cross linker, conjugated PEG-hexanethiol (PEG-C6) with combretastatin A-4 (CA4) to form PEG-C6-AZO-CA4 amphiphilic molecule. These PEG-C6-AZO-CA4 molecules self-assemble into micelles, which can encapsulate hydrophobic anticancer drug. The drug release behavior from PEG-C6-AZO-CA4 micelles was studied under normoxic or hypoxic conditions and the combinations of CA4 with hydrophobic drugs for tumor treatment in vitro were also investigated. As the first example of using AZO linkages to develop anticancer prodrug micelles as hydrophobic anticancer drugs delivery to kill the hypoxic tumor cells in a combination way, this study establishes PEG-C6-AZO-CA4 micelles as a promising drug delivery platform for hypoxic tumor therapy. This journal is

Full text of DOI:10.1039/c4ra14875d

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Development of hypoxia-triggered prodrug
micelles as doxorubicin carriers for tumor therapy†
Cite this: RSC Adv., 2015, 5, 20848
bc
bd
a
bc
e
Hongmei Liu,‡ Ruilong Zhang,‡ Yunwei Niu, Yan Li, Chenmeng Qiao,
e
f
f
a
ab
Jie Weng, Jun Li, Xiaoning Zhang,* Zuobing Xiao* and Xin Zhang*
Received 19th November 2014
Accepted 23rd January 2015
DOI: 10.1039/c4ra14875d
www.rsc.org/advances
Hypoxia has a major role in tumor development and resistance to In some tumors, the tumor tissues partial pressures of oxygen
therapy. Therefore, the effective targeting and killing of hypoxic tumor are near zero mm Hg, compared with the normal tissues
5
cells is a key to successful tumor control. Here, we report the hypoxia- (ꢀ30 mm Hg). Clinical studies have demonstrated that patients
responsive prodrug micelles to deliver hydrophobic anticancer drug, with hypoxic tumors (#10 mm Hg) have signicantly low
6,7
which can selectively release the drugs to treat hypoxic tumor cells in a survival rate. This is because hypoxic tumors are oen resis-
8
combined way. For this purpose, an azobenzene (AZO) bond, which tant to conventional cancer therapies. Therefore, effective tar-
imparts hypoxia sensitivity and specificity as cross linker, conjugated geting and killing of hypoxic cells is a key to successful tumor
PEG–hexanethiol (PEG–C6) with combretastatin A-4 (CA4) to form control. For example, bioreductive prodrugs, such as SN29428,
PEG–C6–AZO–CA4 amphiphilic molecule. These PEG–C6–AZO–CA4 TH302, and SN29966, had been developed for hypoxic tumor
9–12
molecules self-assemble into micelles, which can encapsulate treatment.
However, most of bioreductive prodrugs generate
hydrophobic anticancer drug. The drug release behavior from DNA-reactive cytotoxins, making them difficult to combine with
PEG–C6–AZO–CA4 micelles was studied under normoxic or hypoxic conventional chemotherapy because of overlapping toxicity. On
conditions and the combinations of CA4 with hydrophobic drugs for the other hand, bioreductive prodrugs are confronted with
tumor treatment in vitro were also investigated. As the first example of substantial challenge in drug penetration into hypoxic zones.
using AZO linkages to develop anticancer prodrug micelles as hydro- The penetration problem is particularly severe and has largely
phobic anticancer drugs delivery to kill the hypoxic tumor cells in a been ignored for bioreductive prodrugs, many of which are
2
combination way, this study establishes PEG–C6–AZO–CA4 micelles designed to be metabolized as they diffuse into hypoxic zones.
as a promising drug delivery platform for hypoxic tumor therapy.
Nanoparticles with different materials can deliver drugs with
different mechanisms to tumor and be preferential accumula-
tion in tumor tissues via the enhanced permeation and retention
13,14
(EPR) effect.
However, it is difficult to develop nanoparticle
that can release drugs selectively under hypoxia. Therefore, a
smart hypoxia-responsive linkage was employed for the release
1
. Introduction
Hypoxia is a salient feature of solid tumors and the levels of of drugs under hypoxia, which can contribute signicantly to the
1
–4
hypoxia are more severe in most tumors than normal tissues.
successful treatment of the hypoxic tumor cells.
Most of the hypoxia-sensitive moiety contains nitroaryl or a
15–21
a
quinone group.
Recently, an AZO group was reported that
School of Perfume and Aroma Technology, Shanghai Institute of Technology,
Shanghai, 200233, China. E-mail: xzb@sit.edu.cn; xzhang@ipe.ac.cn
has excellent properties as the hypoxia-sensitive moiety and was
b
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, widely used in the development of imaging agents and bio-
Chinese Academy of Sciences, Beijing, 100190, China
22,23
reductive prodrugs for selectively hypoxic tumors.
Kenjiro
c
University of Chinese Academy of Sciences, Beijing, 100049, China
Hanaoka and co-workers developed AZO-based uorescent
d
Institute of Materials Science and Engineering, Ocean University of China, Qingdao,
probes that made use of the reduction of an AZO group in the
Shandong Province, 266100, China
24
e
reductive environment of hypoxia. Alan C. Sartorelli and co-
Key Laboratory of Advanced Technologies of Materials, School of Materials Science
6
workers synthesized hypoxia-selective O -alkylguanine DNA
and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, 610031, China
f
Collaborative Innovation Center for Biotherapy, School of Medicine, Tsinghua alkyltransferase inhibitors by AZO linkage, which inhibited the
University, Beijing 100084, China. E-mail: drugman@tsinghua.edu.cn
survival of DU145 cells effectively by the combination of lar-
omustine. Currently, most researchers conjugated an AZO
moiety directly to a uorophore to quench the uorescence or to
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra14875d
25
H.L. and R.Z. contributed equally on this manuscript.
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chemical drug to target hypoxic tumor environment. However, when they were accumulated to hypoxic zone of tumor, (3) form
there are no studies available for targeted drug delivery systems stable and compact structure to enhance the colloidal stability. To
using AZO cross linked hypoxia-responsive prodrug micelles.
test this hypothesis, encapsulated hydrophobic anticancer drug
Herein, we have successfully developed AZO-based micelles for (e.g., DOX) PEG–C6–AZO–CA4 micelles (PEG–C6–AZO–CA4/DOX
hydrophobic anticancer drug delivery to kill hypoxic tumor cells micelles) were prepared. The drug release behaviors of
in a combined way and overcome the abovementioned difficulties PEG–C6–AZO–CA4/DOX micelles were evaluated under normoxic
of killing hypoxic tumor cells (Scheme 1). CA4 was conjugated to a or hypoxic conditions. In addition, we examined the DOX bio-
PEG–C6 through an AZO linker to form PEG–C6–AZO–CA4 distribution and synergistic therapeutic effects in MCF-7 cells.
amphiphilic molecules. These PEG–C6–AZO–CA4 amphiphilic Therefore, it is important for the developed hypoxia-sensitive
molecules can self-assemble into AZO-based micelles in an prodrug carrier to co-deliver conventional chemotherapy to cure
aqueous solution, which was named PEG–C6–AZO–CA4 micelles. hypoxic tumor in combination ways.
Due to the introduction of AZO linkage in PEG–C6–AZO–CA4
micelles, it was expected to disassemble the micelles under the
reductive environment of hypoxia. The PEG–C6–AZO–CA4
amphiphilic molecules are composed of four components: (1)
2
. Experimental
2.1. Materials
0
PEG part, (2) C6 part, (3) AZO bond and (4) CA4 part. According to 4,4 -Azobenzenedicarboxylic acid, oxalyl chloride and 1,6-hex-
our design, PEG as hydrophilic part was expected to prolong anedithiol were purchased from J&K Scientic. Triethylamine
PEG–C6–AZO–CA4 micelles circulation time and escape was purchased from Alfa Aesar. DOX$HCl and combretastatin
26
from immunological recognition. Hydrophobic C6 was chosen A-4 (CA4) were obtained from Dalian Meilun Biotech Co. Ltd.,
as a crosslinker to increase the hydrophobic proportion of (Dalian, China). Hoechst 33258, catalase and glucose oxidase
PEG–C6–AZO–CA4. Hydrophobic AZO bond was used because of from Aspergillus niger were obtained from TCI Shanghai.
22–25
its reductive cleavage.
We hoped that when the AZO bond is PEG-MAL2000 was obtained from Shanghai Yare Biotech.
cleaved, the proportion of hydrophobic and hydrophilic micelles Dichloromethane was dried by calcium hydride. Doxorubicin
is changed, resulting in the disassembly of micelles to release the hydrochloride (DOX$HCl) was deprotonated with triethylamine
embedded drugs faster. CA4 is one of the most important natural in dimethyl sulphoxide (DMSO) to obtain the hydrophobic DOX.
molecules that strongly inhibits tubulin polymerization to kill the
27–29
tumor cells.
We chose anticancer drug CA4 for a prodrug 2.2. Synthesis of C6–AZO–CA4
design because of its simple structure with a phenolic group,
hydrophobic feature and its potent cytotoxicity.
0
4
,4 -Azobenzenedicarboxylic acid (100 mg, 0.37 mmol) was
dissolved in anhydrous CH Cl by adding a drop anhydrous
DMF. Oxalyl chloride (125 mL) was dissolved in 10 mL of
anhydrous CH
azobenzenedicarboxylic acid solution. The reaction was carried
out for 2 h at room temperature. The resultant solution was
dried under vacuum. The obtained product was 4,4 -
2
2
Considering the components of PEG–C6–AZO–CA4 mole-
cules, we hypothesized that self-assembled PEG–C6–AZO–CA4
micelles might exhibit the following advantages: (1) encapsulate
hydrophobic drugs (e.g., doxorubicin (DOX)) to kill tumors in
combined effects, (2) release CA4 and hydrophobic drug rapidly
0
2
Cl
2
and was added dropwisely into the 4,4 -
0
Scheme 1 The chemical structure of PEG–C6–AZO–CA4 molecule and schematic illustration of PEG–C6–AZO–CA4/DOX micelles as co-
delivery platform for CA4 and DOX inhibiting the tumor growth in a combined way. Under hypoxic tumor cells, the PEG–C6–AZO–CA4/DOX
micelles release CA4 and DOX rapidly. Released CA4 inhibits tubulin polymerization and the released DOX enters into the nucleus to kill cancer
cells.
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dichloroformylazobenzene. Then, CA4 (117 mg, 0.37 mmol) and 2.7. CA4 equivalent loading, DOX loading efficiency and in
pyridine (87 mL, 1.11 mmol) were dissolved in 10 mL of anhy- vitro release of CA4 and DOX from PEG–C6–AZO–CA4/DOX
0
drous CH Cl and was added dropwisely into 4,4 -dichloro- micelles
2
2
formylazobenzene solution for 1 h. 1,6-Hexanedithiol (59 mL,
The weight of loaded DOX in micelles was measured by a
microplate reader (SpectraMax M5, Molecular Devices, CA)
with excitation at 470 nm and emission at 590 nm aer the
micelles were dissolved in DMSO. The total weight of
PEG–C6–AZO–CA4/DOX micelles was calculated by adding the
weight of PEG–C6–AZO–CA4 molecules used for the prepara-
tion of the micelles and the weight of loaded DOX. The
loading efficiency and loading content of DOX were calculated
0
1
.37 mmol) and pyridine (87 mL, 1.11 mmol) were dissolved in
0 mL of anhydrous CH
2
Cl
2
and was added dropwisely into a
0
solution of CA4-4 and 4 -dichloroformylazobenzene for 2 h. The
crude product was puried by column chromatography on silica
gel (200–300 mesh) eluted with 0–10% methanol gradient in
dichloromethane, which gave C6–AZO–CA4 as a red solid.
3
0
as follows:
2
.3. Synthesis of PEG–C6–AZO–CA4
C6–AZO–CA4 (100 mg, 0.143 mmol) and mPEG-Mal (286 mg,
.143 mmol) were dissolved in anhydrous CH Cl with gentle
weight of loaded DOX
weight of DOX in feed
Loading efficiency ¼
0
2
2
1
stirring for 4 h at room temperature. H NMR (600 MHz, CDCl
3
d ppm) was carried out to characterize the PEG–C6–AZO–CA4.
Loading content ¼
weight of loaded DOX
2
.4. Preparation of PEG–C6–AZO–CA4 micelles and PEG–C6–
weight of PEG ꢁ C6 ꢁ AZO ꢁ CA4=DOX micelles
AZO–CA4/DOX micelles aqueous solution
The PEG–C6–AZO–CA4 molecules (4 mg) were dissolved in
DMSO (200 mL) and then added dropwisely into 1 mL of
deionized water under strong stirring. This solution was then
dialyzed against deionized water for 1 day to remove DMSO. The
PEG–C6–AZO–CA4 molecules (4 mg) and DOX (300 mg) were
dissolved in DMSO (200 mL) and then added dropwisely into
The weight of CA4 in micelles was calculated by the relative
molecular weight between CA4 and PEG–C6–AZO–CA4 mole-
cules. The loading efficiency of CA4 was calculated using the
31
following equation:
Loading content ¼
1
mL of deionized water under vigorous stirring. This solution
weight of CA4 in PEG ꢁ C6 ꢁ AZO ꢁ CA4=DOX micelles
weight of PEG ꢁ C6 ꢁ AZO ꢁ CA4=DOX micelles
was then dialyzed against deionized water for 1 day to remove
DMSO.
The loading content of CA4 into the PEG–C6–AZO–CA4/DOX
micelles was found to be 10.9 wt%. The loading efficiency and
loading content of DOX into the PEG–C6–AZO–CA4/DOX
micelles was found to be 95.2% and 6.7 wt%, respectively.
The release proles of DOX from PEG–C6–AZO–CA4/DOX
2
.5. The morphologies of PEG–C6–AZO–CA4 micelles, PEG–
C6–AZO–CA4/DOX micelles, disassembled PEG–C6–AZO–CA4
micelles and PEG–C6–AZO–CA4/DOX micelles
The morphologies of the micelles were observed by cryoelectron
microscopy (FEI Tecnai 20 cryo-TEM).
micelles were studied using a dialysis tube (MWCO 10 000)
ꢂ
To examine whether these micelles can disassemble in hypoxia under shaking (200 rpm) at 37 C in PBS (pH 7.4). The amount
biological systems, we rst conducted an in vitro assay using rat of DOX released was determined by a microplate reader (Spec-
liver microsomes, which contain various reductases. Aer adding traMax M5, Molecular Devices, CA) with excitation at 470 nm
NADPH (50 mM) as a cofactor for the reductases to rat liver and emission at 590 nm.
ꢁ1
microsomes (75 mg mL ), which containing the aqueous solution
of PEG–C6–AZO–CA4 micelles or PEG–C6–AZO–CA4/DOX micelles,
the morphology of the disassembled micelles was observed by
24
cryo-TEM.
2.8. Enzymatic micelle disassembly process
To examine whether these micelles can disassemble in hypoxia
biological systems, we prepared PEG–C6–AZO–CA4 micelles
embedded hydrophobic DOX. The hydrophobic DOX is released
and gradually subsided when the micelles were disassembled in
2
.6. Size, zeta potential and colloidal stability of micelle
measurements
Micellar size and zeta potential were determined using a Mal- simulation hypoxic environment. The simulation hypoxic envi-
ꢁ1
vern Zetasizer NanoZS. Colloidal stability was measured by ronment was constructed using rat liver microsomes (75 mg mL
)
incubating PEG–C6–AZO–CA4/DOX micelles in Dulbecco's as a reductase and NADPH (50 mM) as a cofactor to consume the
modied Eagle's medium (DMEM) with high glucose supple- oxygen. Aer adding the rat liver microsomes and NADPH mixture
ꢂ
mented with 10% fetal bovine serum (FBS) at 37 C under gentle to the PEG–C6–AZO–CA4/DOX micelles solution, the oxygen
stirring. At each time point, the mean diameters of micelles content was depleted rapidly to near zero in approximately 3 min,
were monitored by dynamic light scattering (DLS).
whereas the oxygen content was about 21% in the normoxic
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32
condition. Aer few minutes, these solutions were imaged by integration values of C6–AZO–CA4, as indicated in Fig. 1B
1
camera.
( H NMR 600 MHz, CDCl d ppm) was carried out to charac-
3
ꢁ1
1
The rat liver microsomes (75 mg mL ) and NADPH (50 mM) terize the C6–AZO–CA4 (46.5 mg, 17.09%). H NMR spectros-
were mixed and added to PEG–C6–AZO–CA4 micelles solution to copy (600 MHz, CDCl d ppm) was carried out to characterize
3
cleave AZO bond. Then, the disassembled PEG–C6–AZO–CA4 the C6–AZO–CA4: 1.34 (–SH, t, J ¼ 7.7 Hz, 1H), 1.46–1.47 (HS–
micelles solution was freeze-dried and dissolved in DMSO. UV-vis CH
absorption spectrum of PEG–C6–AZO–CA4 molecule and cleavage m, 2H), 1.71–1.73 (CH
AZO bond of PEG–C6–AZO–CA4 molecule, which were dissolved (HS–CH –CH
2
–CH
2
–(CH
2
)
2
–CH
2
–, m, 4H), 1.63–1.66 (HS–CH
–CH –CH
, dt, J ¼ 7.3 Hz, 7.5 Hz, 2H), 3.10–3.13 (CH
in DMSO, was measured by UV-vis spectrophotometer (TU1810). S–C]O, t, J ¼ 7.3 Hz, 2H), 3.73 (Ph–O–CH , s, 6H), 3.81
Ph–O–CH , s, 3H), 3.82 (Ph–O–CH , s, 3H), 6.46–6.54 (Ph–CH]
2 2 2
–CH –CH –,
2
2
2
–S–C]O, m, 2H), 2.50–2.56
2
–CH –
2
2
2
3
(
3
3
CH–Ph, m, 2H), 6.54 (CH O–Ph(a,e)–H, s, 2H), 6.91–6.92 (CH O–
2.9. Enzymatic micelles disassembly process in MCF-7 cells
3
3
Ph(i)–H, d, J ¼ 8.5 Hz, 1H), 7.15 (O]C–O–CH O–Ph(k)–H, d, J ¼
3
PEG–C6–AZO–CA4 micelles embedding DOX was prepared as
1
8
8
.8 Hz, 1H), 7.18–7.19 (Ph(h)–H, dd, J ¼ 1.8 Hz, 8.5 Hz, 1H), 8.01–
.03 (–Ph(n,o,p,q)–H, m, 4H), 8.13–8.14 (–Ph(r,s)–H, m, 2H), 8.32–
.33 (–Ph(l,m)–H, m, 2H). The mass and molecular formula of
4
described above. MCF-7 cells (4 ꢃ 10 cells per well) were
seeded in a 35 mm glass bottom culture dish, incubated for 24 h
ꢂ
at 37 C in 5% CO
2
, and allowed to grow until 50% conuent. By
C6–AZO–CA4 were determined by Fourier transform ion cyclo-
tron resonance mass spectrometer (positive, Bruker, USA) m/z
adding PEG–C6–AZO–CA4 micelles to 35 mm glass bottom
culture dish under normoxic or hypoxic, the cells on culture
dish were observed by live cell station. The condition of hypoxia
in cells was generated by the direct depletion of oxygen in sealed
environment using the glucose oxidase (2 units per mL) and
catalase (120 units per mL) dual enzyme system as previously
+
+
7
01.23 [M + H] , 718.23 [M + NH ] ; calcd for C H N O S :
4 38 40 2 2 2
700.23, found: 701.23 (Fig. S1†). As shown in Fig. S1,† the result
was consistent with the expected formula of the C6–AZO–CA4.
These data indicated that C6–AZO–CA4 was synthesized. The
C6–AZO–CA4 was then coupled with PEG-Mal2000 to form
PEG–C6–AZO–CA4 molecule. The formation of PEG–C6–AZO–CA4
32
described. All images were collected with UltraVIEW® VoX 3D
live cell imaging system (PerkinElmer). For time-lapse experi-
ments, images were collected every 30 s for about 60 min.
To investigate the intracellular drug release from micelles, a
1
was rst conrmed by H-NMR spectroscopy. The reaction at the
maleimide group of PEG was conrmed by the disappearance of
double bond of PEG-Mal at d 6.94 ppm and the appearance the B
position of PEG–C6–AZO–CA4 at d 3.47 ppm. The PEG peak of
PEG–C6–AZO–CA4 molecule appeared at d 3.64 ppm (Fig. 1C).
5
density of 1 ꢃ 10 MCF-7 cells were plated into 35 mm Petri dish
aer 24 hours incubation, and then, the cells were incubated
with micelles under normoxic or hypoxic conditions for 4 h. For
nuclear staining, the cells were incubated with Hoechst 33285
ꢂ
for 10 min at 37 C and then washed by PBS (pH 7.4) twice. The
3.2. Preparation and characterization of PEG–C6–AZO–CA4
intracellular localization of DOX released from micelles was
observed by confocal laser scanning microscopy (CLSM).
micelles
Aer the successful synthesis of the PEG–C6–AZO–CA4 mole-
cules, self-assembly and encapsulating hydrophobic drugs
abilities of PEG–C6–AZO–CA4 were evaluated. In this study,
2.10. Drug toxicity studies
Clonogenic survival assays were performed as described previ- DOX was chosen as model anticancer drug with two features.
3
2
2
ously. MCF-7 cells were plated into plastic 25 cm tissue culture First, DOX causes DNA damage and the induction of apoptosis

5
asks at a density of 5 ꢃ 10 cells and grown for 24 h. The cells by the inhibition of the progression of the enzyme top-
33
were pretreated for 1 h without drug in the presence of glucose oisomerase II. CA4 is one of the most important natural
oxidase (2 units per mL), catalase (120 units per mL) and 10 mM molecules that strongly inhibit tubulin polymerization.
of added glucose to generate hypoxic condition. Aer pretreat- Previous research has shown that combined DOX with CA4 to
3
4,35
ment, PEG–C6–AZO–CA4 micelles or PEG–C6–AZO–CA4/DOX cure tumor got effective results.
In this study, we used
micelles with two different concentrations were added, and the PEG–C6–AZO–CA4 micelles to encapsulate DOX to cure
treatment continued for an additional 2 h. Aer treatment, the hypoxic tumor in a combined way. Second, DOX had auto-
cells were detached by trypsinization, suspended in culture uorescence, which was easy to reveal micelles' properties
medium and counted. Cells were plated into six-well plates at a and the interaction between the drug and micelle. The
density of 1000 cells per well. Seven days later, colonies were PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX

xed, stained with crystal violet, and counted.
micelles were prepared and characterized by cryo-TEM and DLS.
As shown in Fig. 2A and B, PEG–C6–AZO–CA4 micelles and
PEG–C6–AZO–CA4/DOX micelles were spherical micelles with
unimodal size distribution by cryo-TEM. An average diameter of
PEG–C6–AZO–CA4 micelles was about 65.9 ꢄ 4.3 nm in an aqueous
3
. Results and discussion
3.1. Synthesis and characterization of PEG–C6–AZO–CA4
To test the aforementioned hypotheses, the PEG–C6–AZO–CA4 solution, which was determined by DLS studies. PEG–C6–AZO–CA4
molecule was synthesized. The synthetic routes and chemical micelles encapsulated DOX with a particle size of 75.9 ꢄ 4.9 nm
structures of PEG–C6–AZO–CA4 are given in Fig. 1A. The were bigger than PEG–C6–AZO–CA4 micelles because they were
1
formation of C6–AZO–CA4 was rst conrmed by H-NMR embedded with DOX (Fig. 2C). The particle size of micelles (20–100
spectroscopy with all the characteristic peaks and the nm) was benecial for tumor targeting delivery, which was large
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1
3
Fig. 1 Synthesis of PEG–C6–AZO–CA4. (A) Synthetic route of the PEG–C6–AZO–CA4. (B) H NMR spectra of C6–AZO–CA4 (600 MHz, CDCl ).
1
(
3
C) H NMR spectra of PEG–C6–AZO–CA4 (600 MHz, CDCl ).
enough to avoid renal ltration and lymphatic clearance and of hypoxia, we rst prepared PEG–C6–AZO–CA4 micelles and
small enough to penetrate through the leaky vasculatures in PEG–C6–AZO–CA4/DOX micelles and conducted an in vitro assay
3
6
tumor region. Therefore, PEG–C6–AZO–CA4/DOX micelles in rat liver microsomes, which contain various reductases, mixed
24
were easy to accumulate in tumor region. The equivalent CA4 with NADPH. Aer adding NADPH (50 mM) as a cofactor for the
loading efficiency of PEG–C6–AZO–CA4/DOX micelles was reductases to yellow PEG–C6–AZO–CA4 prodrug micelles aqueous
ꢁ
1
1
0.9 wt%. The loading efficiency and loading content of DOX solution (4 mg mL ) in the presence of rat liver microsomes
ꢁ
1
into the PEG–C6–AZO–CA4/DOX micelles was found to be (75 mg mL ) (Fig. 2Da1), the yellow solution slowly turned color-
5.2% and 6.7 wt%, respectively. The PEG–C6–AZO–CA4/DOX less and a yellow solid appeared in centrifuge tube (Fig. 2Da2).
micelles reduced the amount of inert materials to embed Moreover, the red PEG–C6–AZO–CA4/DOX micelles aqueous
9
ꢁ
1
DOX and enhanced the CA4 loading efficiency due to conju- solution (4 mg mL ) (Fig. 2Db1) slowly turned transparent
gated CA4 as a component of micelle. and the red precipitate was present (Fig. 2Db2) under
Due to the introduction of hypoxia-sensitive AZO bonds, the the same hypoxic condition with PEG–C6–AZO–CA4 micelles.
PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles These results suggested that the micelles solution under
were expected to disassemble quickly when they are exposed to reductive hypoxic condition resulted in the cleavage of the AZO
reductive enzymes under the conditions of hypoxia. To examine bonds and released the drugs. To further conrm the disassembly
whether PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX of micelles under reductive hypoxic condition, the morphologies
micelles can disassemble by reductive enzymes under conditions of PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX
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Fig. 2 The cryo-TEM image of PEG–C6–AZO–CA4 micelles. (B) The cryo-TEM image of PEG–C6–AZO–CA4/DOX micelles. Bar ¼ 200 nm. (C)
The size distribution of PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles by DLS. (D) (a1) The image of PEG–C6–AZO–CA4
micelles; (a2) the image of disassembled PEG–C6–AZO–CA4 micelles. (b1) The image of PEG–C6–AZO–CA4/DOX micelles; (b2) the image of
disassembled PEG–C6–AZO–CA4/DOX micelles. (E) The cryo-TEM image of disassembled PEG–C6–AZO–CA4 micelles. (F) The cryo-TEM
image of disassembled PEG–C6–AZO–CA4/DOX micelles. Bar ¼ 200 nm. (G) The size distribution of PEG–C6–AZO–CA4 micelles and
PEG–C6–AZO–CA4 micelles after the enzyme treatment for 2 min. (H) The size distribution of PEG–C6–AZO–CA4/DOX micelles and
PEG–C6–AZO–CA4/DOX micelles after the enzyme treatment for 2 min. (I) The cleavage of the AZO bond mechanism of PEG–C6–AZO–CA4
molecule. (J) UV-vis absorption spectrum of PEG–C6–AZO–CA4 molecule (red line) and cleavage AZO bond of PEG–C6–AZO–CA4 molecule
ꢂ
(
blue line) in DMSO at 25 C.
micelles aer treatment with NADPH and rat liver microsomes gradually became bigger, some micelles disassembled and the
were observed by cryo-TEM. From Fig. 2E and F, the size of number of micelles decreased. DLS analysis further conrmed
PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles these conclusions that the size and the polydispersity index (PDI)
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Fig. 3 (A) The stability of PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles in DMEM with 10% FBS (n ¼ 3). (B) Release
profiles of DOX from PEG–C6–AZO–CA4/DOX micelles (n ¼ 3).
of PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles and PEG–C6–AZO–CA4/DOX micelles to living tumor
micelles became bigger aer the enzyme treatment for 2 min cells (MCF-7). Nano-environment induces self-organization
(
Fig. 2G and H). The counting rate of PEG–C6–AZO–CA4 micelles of DOX molecules driven by their intermolecular p–p stacking
26,39–41
and PEG–C6–AZO–CA4/DOX micelles were decreased from 350 to interaction.
Therefore, when the DOX encapsulated
119 and from 399 to 149, respectively. These results coincided into the micelles, the uorescent signal of DOX molecules
with the cryo-TEM observed and showed that the number of is quenched almost completely. Once released from the
micelles was decreased. The cleavage of AZO bond mechanism of micelles, the uorescence of DOX molecules is restored. We
PEG–C6–AZO–CA4 molecule is shown in Fig. 2I. AZO bond used this photophysical property of DOX to investigate the
conjugated with different groups will lead to a signicant red shi hypoxia-responsive activated drug release performance of
37
of the p–p* band. As shown in Fig. 2J, PEG–C6–AZO–CA4 PEG–C6–AZO–CA4/DOX micelles. The release behavior of DOX
molecule had a broad absorption band centered at 450 nm, from PEG–C6–AZO–CA4/DOX micelles was observed using
which is typical for the AZO chromophore. This absorption range UltraVIEW® VoX 3D live cell imaging system. Almost no DOX
disappeared when the AZO bond of PEG–C6–AZO–CA4 uorescence was observed in MCF-7 cells following 60 min
ꢁ
1
molecule was cleaved. Therefore, PEG–C6–AZO–CA4 micelles and incubation with PEG–C6–AZO–CA4/DOX micelles (2 mg mL
)
PEG–C6–AZO–CA4/DOX micelles were disassembled and triggered under normoxic conditions in merged images (Fig. 4Aa and
the fast release of encapsulated DOX and free CA4 under the Video S1 in the ESI†), whereas less DOX uorescence was
conditions of reductive enzymes in hypoxia.
observed following 20 min, and at 60 min, the strongest DOX
The in vitro colloidal stability of PEG–C6–AZO–CA4 micelles and uorescence appeared in merged image under hypoxic condi-
PEG–C6–AZO–CA4/DOX micelles was investigated in DMEM con- tions (Fig. 4Ab and Video S2 in the ESI†). These results
taining 10% FBS. As shown in Fig. 3A, for 72 hours incubation of indicate that PEG–C6–AZO–CA4/DOX micelles release DOX
PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX micelles, rapidly and indicate that AZO bonds are cleaved under hypoxic
no signicant size changes were observed. These results suggested conditions just for a few minutes. Aer the incubation of
that PEG–C6–AZO–CA4 micelles and PEG–C6–AZO–CA4/DOX PEG–C6–AZO–CA4/DOX micelles for 4 h under normoxic
micelles had better colloidal stability in FBS. The release of condition in MCF-7 cells, less uorescence appeared in cytoplasm
DOX from PEG–C6–AZO–CA4/DOX micelles was studied in PBS and there was no DOX uorescence in nuclei (Fig. 4Ba). Moreover,
ꢂ
(
pH 7.4) at 37 C. The result showed that 12.2% of DOX was free DOX was trafficked to the cell nuclei and strong DOX uo-
released in 4 days at pH 7.4 PBS (Fig. 3B). This result indicated that rescence appeared in perinuclear under hypoxic condition
PEG–C6–AZO–CA4/DOX micelles are sufficiently stable under (Fig. 4Bb). These ndings are in good agreement with UltraVIEW®
physiological conditions, whereas the red solution turned colorless VoX 3D live cell imaging system results. Under normoxic condition,
and a red solid appeared in the presence of double enzyme, as DOX release from PEG–C6–AZO–CA4/DOX micelles through
42
shown in the Fig. 2Db. Therefore, these ndings are important hydrolysis of the ester linkage between CA4 and PEG–C6–AZO.
because colloidal stability in FBS and slow release of the drug from From the UltraVIEW® VoX 3D live cell imaging system and CLSM
micelles in physiological environment for any nanoparticles (NPs) results, we found that PEG–C6–AZO–CA4/DOX micelles tend to
determines the successful delivery of drugs to target position as it passively release DOX and are limited to release an effective drug
prevents particle aggregation or embolism and premature burst concentration at desired time and anti-tumor efficacy under nor-
drug release from circulatory system and prolongs the NPs resi- moxic condition. This was likely due to the retarded hydrolysis
38
43
dence time in the body.
and drug diffusion within the hydrophobic core. Moreover,
PEG–C6–AZO–CA4/DOX micelles released DOX quickly and
reached maximum tumor accumulation at the time point under
hypoxic condition, which signicantly improved therapeutic effects
in hypoxic tumor. Overall, PEG–C6–AZO–CA4/DOX micelles were
highly susceptible to hypoxic conditions and released DOX rapidly.
3
.3. Characterization of PEG–C6–AZO–CA4 micelles in
hypoxic tumor cells
In order to examine the release proles of micelles under nor-
moxic or hypoxic tumor cells, we applied PEG–C6–AZO–CA4
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Fig. 4 (A) Images were collected from UltraVIEW® VoX 3D live cell imaging system at indicated times. Original time-lapse imaging was taken
ꢁ1
every 30 s for 60 min in the presence of PEG–C6–AZO–CA4/DOX micelles (0.25 mg mL ) containing 16 mg DOX under normoxic (a) or hypoxic
ꢁ1
condition (b). Bar ¼ 12 mm. (B) CLSM images of PEG–C6–AZO–CA4/DOX micelles (0.25 mg mL ) containing 16 mg DOX under normoxic (a) or
hypoxic (b) conditions in live MCF-7 cells for 4 h. Bar ¼ 25 mm. The excitation and emission wavelengths were 488 nm and 590 nm for DOX,
respectively. (C) Images were collected from UltraVIEW® VoX 3D live cell imaging system at indicated times. Original time-lapse imaging was
ꢁ1
taken every 30 s for 60 min in the presence of PEG–C6–AZO–CA4 prodrug micelles (0.25 mg mL ) under normoxic (a) or hypoxic conditions
b). Bar ¼ 12 mm (red arrows indicate thin retraction fibers and white arrows indicate distance between the cells).
(
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they are under normoxic condition. Compared with the cyto-
toxicity of PEG–C6–AZO–CA4 micelles with CA4, the cytotoxicity
of PEG–C6–AZO–CA4/DOX micelles was considerably lower, no
matter what the condition was, due to a synergistic action of
DOX and free CA4. Notably, the survival of MCF-7 cells was
lowest aer treatment with PEG–C6–AZO–CA4 micelles
embedded DOX (30 mM equivalent CA4 concentration and
4
0
.5 mM DOX concentration), which reduced viability by 7.6ꢄ
.68% compared to control under hypoxic condition (Fig. 5).
This observation was consistent with our ndings on releasing
DOX and free CA4 quickly under hypoxic tumor cells, which
enhanced the anticancer efficacy.
4. Conclusions
In summary, we developed AZO-based prodrug micelles
PEG–C6–AZO–CA4 micelles) for DOX delivery. AZO-based pro-
(
drug micelles have a built-in hypoxic trigger release mechanism
to further enhance drug deposition in tumors and enhanced the
anticancer activity. We have successfully demonstrated the
PEG–C6–AZO–CA4 micelles released DOX and free CA4 quickly
under hypoxic conditions. Moreover, the strong cytotoxicity of
CA4 and DOX was examined in MCF-7 cells. This nding sug-
gested that the PEG–C6–AZO–CA4 micelles had the potential for
the delivery of two drugs to provide a promising nanomedicinal
approach for cancer treatment.
Fig. 5 Cytotoxicity of PEG–C6–AZO–CA4 micelles and PEG–C6–
AZO–CA4/DOX micelles under normoxic and hypoxic conditions. The
vertical axis indicates the percentage of survival. The horizontal axis
indicates the concentration of DOX and CA4 equivalent concentra-
tions. Means ꢄ SD, n ¼ 3.
To evaluate the CA4 release behavior of PEG–C6–AZO–CA4
micelles under normoxic or hypoxic conditions in MCF-7 cells,
we performed time-lapse imaging of MCF-7 cells in micelles for
Acknowledgements
6
0 min. CA4, isolated from Combretum caffrum, binds to the
This work was nancially supported by the National Natural
Science Foundation of China (Grants no. 21304099, 51203162,
44,45
colchicines binding site of b-tubulin.
most important natural molecules that strongly inhibits tubulin
polymerization to kill the tumor cells. Some previous studies
described that cells treated with CA4 would retract the cell
margins, formed the thin retraction bers and caused loss of
cell–cell interaction.
cells contraction, formed thin retraction bers (red arrow) and
caused loss of cell–cell interaction (white arrow) starting within
CA4 is also one of the
5
1103159, 51373177), the National High Technology Research
and Development Program (Grants no. 2014AA020708,
012AA022703, 2012AA020804), the Instrument Developing
10
2
Project of the Chinese Academy of Sciences (Grant no.
YZ201253, YZ201313), Open Funding Project of the National
Key Laboratory of Biochemical Engineering (Grant no.
Y22504A169). “Strategic Priority Research Program” of the
Chinese Academy of Sciences, Grant no. XDA09030301-3 was
also acknowledged.
46,47
PEG–C6–AZO–CA4 micelles caused
6
0 min of micelles addition under normoxic conditions
(Fig. 4Ca), it also occurred in Fig. 4A, especially in the Video S1,†
where the phenomenon is obvious. These results suggested that
PEG–C6–AZO–CA4 micelles can release free CA4 to inhibit
tubulin polymerization and release free CA4 quickly under
hypoxic condition, due to the cleavability of AZO bond under
hypoxic condition. These results coincided with the above-
mentioned DOX released behaviors.
Notes and references
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