Reduction-sensitive mixed micelles assembled from amphiphilic prodrugs for self-codelivery of DOX and DTX with synergistic cancer therapy

Article abstract of DOI:10.1016/j.colsurfb.2017.11.011

Clinically, codelivery of chemotherapeutics has been limited by poor water-solubility and severe systemic toxicity. In this work, we developed a new reduction-sensitive mixed micellar system for self-codelivery of doxorubicin (DOX) and docetaxel (DTX). Biodegradable methoxy poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL) was coupled with DOX and DTX by a reduction-sensitive disulfide bond, resulting in mPEG-PCL-SS-DOX and mPEG-PCL-SS-DTX, respectively. mPEG-PCL-SS-DOX was mixed with mPEG-PCL-SS-DTX at a mole ratio of 1:1 in water, forming a mixed micellar system. The mixed micelles had a diameter of 223.7 nm and a low critical micelle concentration. Reductive-triggered drug release revealed a “smart” characteristic of the mixed micelles. A cellular uptake and cytotoxicity assay in vitro showed that the mixed micelles could efficiently accumulate in MCF-7 cells and suppress the growth of tumour cells. The proposed reduction-sensitive mixed micelles assembled from amphiphilic prodrugs can be used as a promising drug codelivery system for cancer therapy.

Full text of DOI:10.1016/j.colsurfb.2017.11.011

Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Full Length Article
Reduction-sensitive mixed micelles assembled from amphiphilic
prodrugs for self-codelivery of DOX and DTX with synergistic cancer
therapy
Jilian Wua, Huiyuan Zhanga, Xu Hua, Ruiling Liua, Wei Jianga, Zhonghao Lib,
Yuxia Luana,∗
a School of Pharmaceutical Science, Key Laboratory of Chemical Biology, Ministry of Education, Shandong University, Jinan, 250012, China
b Key Lab of Colloid & Interface Chemistry, Shandong University, Ministry of Education, Jinan, 250100, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Clinically, codelivery of chemotherapeutics has been limited by poor water-solubility and severe sys-
temic toxicity. In this work, we developed a new reduction-sensitive mixed micellar system for
self-codelivery of doxorubicin (DOX) and docetaxel (DTX). Biodegradable methoxy poly(ethylene glycol)-
poly(␧-caprolactone) (mPEG-PCL) was coupled with DOX and DTX by a reduction-sensitive disulfide
bond, resulting in mPEG-PCL-SS-DOX and mPEG-PCL-SS-DTX, respectively. mPEG-PCL-SS-DOX was
mixed with mPEG-PCL-SS-DTX at a mole ratio of 1:1 in water, forming a mixed micellar system. The
mixed micelles had a diameter of 223.7 nm and a low critical micelle concentration. Reductive-triggered
drug release revealed a “smart” characteristic of the mixed micelles. A cellular uptake and cytotoxicity
assay in vitro showed that the mixed micelles could efficiently accumulate in MCF-7 cells and suppress the
growth of tumour cells. The proposed reduction-sensitive mixed micelles assembled from amphiphilic
prodrugs can be used as a promising drug codelivery system for cancer therapy.
Received 14 June 2017
Received in revised form
29 September 2017
Accepted 4 November 2017
Available online 6 November 2017
Keywords:
polymeric micelles
doxorubicin
docetaxel
reduction-sensitive
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
which the ratio of the mean relative dose intensity for DOX and DTX
is 1:1. Unfortunately, chemotherapeutic drugs have no selectivity
Various combinations of drugs with different mechanisms of
action have become increasingly relevant in cancer treatment as
multi-pronged attacks using different drugs could deter the devel-
opment of drug resistance [1–4]. Doxorubicin (DOX) and docetaxel
(DTX) are the most crucial antitumor drugs used clinically for
cancer treatment, and they are well-known as inhibitors of topoi-
somerase II and microtubule disassembly, respectively [5]. There
have been a few studies combining DOX and DTX for the treatment
of the patients suffering from breast and prostate cancers [6–9].
DOX and DTX possess different mechanisms of action and differ-
ent toxicity profiles and resistance. As a result, the combination of
DOX and DTX can decrease their systemic toxicity and deter the
development of drug resistance by the administration of a lower
concentration of each drug. The therapeutic effect of drug combi-
nations is controlled by the mixing ratios of the drugs. Studies have
shown that a combinatorial regimen consisting of DTX (75 mg/m2)
and DOX (60 mg/m2) is feasible with an effective schedule [10,11] in
and they also kill normal cells in addition to malignant cells, caus-
ing undesired systemic toxicity. In addition, poor water-solubility
and rapid blood/renal clearance also make them difficult to use in
clinical applications.
Drug carriers with a variety of architectures, including poly-
meric micelles [12,13], liposomes [14,15], dendrimers [16], carbon
nanotubes [17], and gold nanoparticles [18], have been explored to
transport drugs in a controlled and targeted fashion. With the help
of nanocarriers, drugs could be effectively delivered and accumu-
lated in tumour tissues by an enhanced permeability and retention
(EPR) effect. Polymeric micelles have a capacity for loading mul-
tiple anticancer drugs, and they act as an injectable multi-drug
nanocarrier by physical loading and chemical loading. Particularly,
chemical crosslinking may be an important strategy that helps to
retain micellar structural integrity and prevent premature leak-
age of drugs for long-term circulation in the blood. However, such
crosslinking could also hamper the desired drug release at the tar-
get site. Stimuli-triggered release is an efficient way to release the
drug in a controlled manner on arrival at the target site. Until now,
various stimuli-responsive nanocarriers have been studied exten-
sively, such as pH, temperature, redox potential or oxidative stress,
∗
Corresponding author.
E-mail address: yuxialuan@sdu.edu.cn (Y. Luan).
https://doi.org/10.1016/j.colsurfb.2017.11.011
0927-7765/© 2017 Elsevier B.V. All rights reserved.

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J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
Scheme 1. Illustration of the sealf-assembly, accumulation in the tumour tissue and intracellular trafficking pathway of reduction-sensitive mPEG-PCL-SS-DOX/mPEG-PCL-
SS-DTX (1:1) mixed micelles.
magnetic field, light, and ultrasound [19]. Among these sensitivi-
ties, reduction-sensitive nanocarriers have attracted considerable
attention and have been widely studied since tumour tissues have
high redox potential (glutathione (GSH), approximately 1–10 mM)
in the cytosol and cell nuclei compared with the plasma (GSH,
approximately 2 ␮M) [20]. Moreover, the tumour tissues showed at
least fourfold higher concentrations of GSH levels than did normal
cells in tumour-bearing mice [21]. Therefore, a common reduction-
sensitive chemical bond, such as a disulfide bond, used as a linker
between drug and polymer could easily be cleaved to release drugs
at tumour sites with the reductive microenvironment.
acid (DTDP), acetyl chloride, 4-dimethylaminopyridine (DMAP),
N, N-dicyclohexylcarbodiimide (DCC), triethylamine (TEA),
N-hydroxysuccinimide (NHS) and dithiothreitol (DTT) were
all obtained from Aladdin Industrial Corporation. N, N-
dimethylformamide (DMF) and dimethyl sulfoxide (DMSO)
were purchased from Sinopharm Chemical Reagent Co., Ltd.
Dichloromethane (DCM) was obtained from Tianjin Fuyu Fine
Chemical Co., Ltd. Acetonitrile was purchased from Shanghai
Siyou Co., Ltd., China. mPEG-PCL-SS-COOH (PCL, Mw = 2000) was
synthesized as described in our previous study.
In our study, we developed a new reduction-sensitive mixed
micellar system assembled from the amphiphilic prodrugs to
realise the self-codelivery of DOX and DTX (Scheme 1). Methoxy
poly(ethylene glycol) (mPEG) was chosen as a hydrophilic block of
polymeric micelles because of its good biocompatibility, stealth-
like nature, and high mobility [22,23]. Poly(␧-caprolactone) (PCL)
was chosen as a hydrophobic block of micelles due to its biodegrad-
ability and low toxicity. Amphiphilic block copolymer mPEG-PCL
was coupled with DOX and DTX by a reduction-sensitive disul-
fide bond, resulting in mPEG-PCL-SS-DOX and mPEG-PCL-SS-DTX,
respectively. mPEG-PCL-SS-DOX was mixed with mPEG-PCL-SS-
DTX at a mole ratio of 1:1 in water, forming mixed micelles by
self-assembly. The resulting mixed micelles were extensively char-
acterized with regard to their morphology, particle size and critical
micelle concentration (CMC). The drugs that were in vitro released
were monitored in the presence and absence of dithiothreitol (DTT).
The in vitro cellular uptake and cytotoxicity of mixed micelles to
MCF-7 breast cancer cells were also investigated. Moreover, the
safety of mixed micelles was evaluated with a haemolytic test
in vitro.
2.2. Synthesis of mPEG-PCL-SS-DOX and mPEG-PCL-SS-DTX
For the synthesis of mPEG-PCL-SS-DOX, the hydrophobic DOX
was obtained by adding TEA to DOX·HCl solution followed by stir-
ring at room temperature for 12 h in the dark. mPEG-PCL-SS-COOH,
NHS and DCC were dissolved in the DCM and stirred in an ice bath
for 24 h to activate the carboxyl groups. The reaction solution was
filtered and the solvent was removed by a rotary evaporator. The
residue derived from above and DMAP was dissolved in DMSO with
DOX and stirred at room temperature for 48 h in the dark. The
resulting solution was dialysed (MWCO 3500 Da) against DMSO for
24 h to remove the residual DOX. DMSO was removed by dialysing
in water. The final product mPEG-PCL-SS-DOX was obtained by
freeze-drying. The synthesis of mPEG-PCL-SS-DTX was carried out
according to our previous work [24].
2.3. Nuclear magnetic resonance spectroscopy (NMR) and Fourier
transform infrared spectroscopy (FTIR) analysis
1H NMR spectroscopy was used for the structural confirma-
tion of mPEG-PCL-SS-DOX or mPEG-PCL-SS-DTX conjugates and
intermediates. All spectra were obtained via an NMR spectrome-
ter (Mercury Plus 400, Varian, USA) using chloroform-d (CDCl3) or
dimethyl sulfoxide-d6 (DMSO-d6) as a solvent. A FTIR spectrome-
ter was employed for characterization of the synthesized prodrug
copolymers. The samples were mixed with potassium bromide
and punched to a tablet for characterization. The FTIR data were
recorded on a Nicolet 6700 Fourier transform infrared spectrome-
ter.
2. Material and methods
2.1. Materials
DTX and doxorubicin hydrochloride (DOX·HCl) were sup-
plied by Dalian Meilun Biotech Co., Ltd. Methoxy poly (ethylene
glycol) (mPEG, Mw = 2000) was obtained from Sigma-Aldrich.
Stannous isooctoate, ␧-caprolactone, 3, 3-dithiodipropionic

J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
451
2.4. Preparation of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1)
mixed micelles
250 mm × 4.60 mm). The mobile phase was acetonitrile: water
(55:45, v/v). The flow rate was set to 1 mL/min with a detection
wavelength of 230 nm.
Based on our previous work [25] about codelivery of Paclitaxel
and DOX, and another group’s work about codelivery of DTX and
DOX [10,11], the optimal synergistic combination of DOX and DTX
(mole ratio = 1:1) was chosen as the system of study. mPEG-PCL-
SS-DOX/mPEG-PCL-SS-DTX (mole ratio = 1:1) mixed micelles were
prepared by the membrane dialysis method [26]. mPEG-PCL-SS-
DOX was mixed with mPEG-PCL-SS-DTX in DMSO at the mole ratio
of 1:1. Then, 4 mL of distilled water was added drop-by-drop. The
formed solution was transferred to a dialysis bag (MWCO 3500 Da)
and dialysed against distilled water for 24 h at room temperature.
2.7. Cellular uptake
The cellular uptake was studied by flow cytometry and fluo-
rescence microscopy imaging. For the flow cytometry analysis to
quantitatively understand the cellular uptake, MCF-7 cells were
seeded at a density of 1 × 105 cells per plate containing 2 mL
of DMEM media with 10% foetal bovine serum at 37 ◦C in a 5%
CO2 atmosphere and allowed to attach for 24 h. Subsequently, the
medium was replaced with the mixed micelles and free DOX with
a DOX concentration of 3 ␮g/mL. After 4 h incubation at 37 ◦C,
the cells were washed with PBS three times and extracted by a
trypsinisation process. The cells were washed two times with PBS
containing heparin again, centrifuged and suspended in a flow
cytometry buffer to measure the fluorescent intensity.
2.5. Characterization of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX
(1:1) mixed micelles
2.5.1. CMC of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) mixed
micelles
The CMC of the mixed micelles was determined using pyrene as
a fluorescence probe [27]. Briefly, micellar aqueous solution was
mixed with pyrene to obtain a series of polymer solutions var-
ied from 9 × 10−8 mM to 9 × 10−2 mM. The final concentration of
pyrene in each sample was fixed at 6 × 10−6 M. Pyrene fluorescence
spectra was measured using a fluorescence spectrophotometer
(F-7000, Hitachi, Japan) with the excitation wavelength fixed at
334 nm. The emission spectra were recorded from 300 nm to
450 nm. The experiment was repeated three times, and the results
presented were expressed as the mean standard derivation (SD).
The ratio of I373/I383 was plotted against the mixed micelle concen-
tration and the CMC value was obtained from the inflection point
of the diagram.
For the fluorescence microscopy imaging to qualitatively under-
stand cellular uptake, after incubation of samples in culture
medium at 37 ◦C for 4 h, the medium was removed and the cells
were rinsed with PBS three times. Then, the cells were observed by
fluorescent inverted microscopy (ECLIPSE-Ti, Nikon).
2.8. In vitro cytotoxicity assay
MCF-7 cell lines were used to evaluate the antitumour activ-
ity of mixed micelles. The cells were seeded in 96-well plates at
a density of 8 × 103 cells/well and incubated in medium with 10%
foetal bovine serum (containing 5% CO2) at 37 ◦C for 24 h. Then,
the medium was removed and the cells were treated with differ-
ent concentrations of the tested sample solutions for 24 h, 48 h and
72 h. The drug concentration ranged from 1 nM to 10000 nM. Next,
the medium was removed and MTT (3-(4, 5-dimethylthiazol-2-yl)-
2, 5-dipheny-ltetrazolium bromide) solution (5 mg/mL in PBS) was
added to each well. Then, the cells were incubated for 4 h. After the
removal of the medium containing MTT, 150 ␮L of DMSO was added
to dissolve the MTT crystals. The optical density of the solution was
measured using a microplate reader (ELIASA of Perkin Elmer) at a
wavelength of 490 nm. All the cytotoxicity tests were conducted
in triplicate, and the data were presented as the mean standard
2.5.2. Particle size and morphology characterization
The average hydrodynamic diameter and polydispersity index
of mixed micelles were obtained by dynamic light scattering (BIC-
Brook-Haven, USA). Polymeric micelles formulation was diluted
with purified water by 1:3 (v/v) before the measurement. The
morphology of prepared micelles was imaged with transmission
electron microscopy (TEM) (JEM-200CX, NEC Company, Japan).
2.6. Drug release in vitro
In vitro release of DOX and DTX from the mPEG-PCL-SS-
DOX/mPEG-PCL-SS-DTX (1:1) mixed micelles was evaluated in the
absence and presence of DTT (10 mM) in PBS (pH 7.4) at 37 ◦C.
Briefly, 1 mL of freshly prepared mixed micelles containing 300 ␮g
DTX and 202 ␮g DOX were loaded into dialysis bag with a MWCO
of 3500 Da. Subsequently, the dialysis bags was immersed in 40 mL
PBS containing 0.5% (w/v) Tween 80 (pH 7.4) in the absence and
presence of DTT (10 mM) and shaken in a 37 ◦C water bath at
100 rpm. 3 mL of release medium was taken out and replaced with
an equal volume of fresh buffer solution at the desired time inter-
vals. The release experiments were carried out in triplicate, and the
data were presented as the mean standard derivation (SD).
derivation (SD). The half maximal inhibitory concentration (IC50
)
representing the concentration of the drug corresponding to 50%
cell inhibition in vitro, was calculated using Statistical Package for
the Social Sciences (SPSS) software.
2.9. Haemolysis test
The haemolytic effect of the formulation was evaluated using
New Zealand rabbit erythrocytes. Briefly, the plasma samples
obtained from rabbits were centrifuged and diluted with normal
saline to separate the red blood cells (RBCs 2%). Afterwards, 1.25 mL
red blood cells were added to each sample. For controls, 1.25 mL
of normal saline (negative controls) and 1.25 mL of distilled water
(positive control) were added. The mixed micelles (0.15 mL) at var-
ious concentrations (0.1, 1, 50, 100 and 200 ␮M) were incubated
with a 1.25 mL RBC suspension, and then normal saline was added
to maintain the same volume. All samples were kept at 37 ◦C for
3 h and then centrifuged at 1500 rpm for 15 min. The absorbance
of the supernatant was measured using a UV spectrophotometer at
576 nm [28]. The studies were done in triplicate and the data were
presented as the mean standard derivation (SD). The haemolysis
DOX
possesses
a
fluorescent
hydroxy-substituted
anthraquinone chromophore. Therefore the concentration of
DOX can be quantified by fluorescence measurements. The con-
centration of DTX was measured by high-performance liquid
chromatography because DTX has no fluorescence characteris-
tics. For example, the concentration of DOX was quantified by
fluorescence measurements (F-7000, Hitachi, Japan) with the
excitation wavelength fixed at 496 nm and the emission wave-
length fixed at 558 nm. The concentration of DTX was measured
by high-performance liquid chromatography (HPLC, Agilent Tech-
nologies) using a Hypersil Gold C18 column (5 ␮m particle size,

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J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
percentage of RBCs was determined using the following equation:
our amphiphilic block copolymer, it is capable of self-assembly to
form micelles. The size of our micelles is much larger than nor-
mal micelles, and we consider three major possibilities could be
responsible: (1) small micelles gather to form large clusters, which
coexist with the regular micelles [32]. (2) PEG chains in aqueous
solution can form a large and loose thermodynamically reversible
association complex. (3) A few free drugs are loaded into micelles
so that the micelle size is increased [33].
Asample − Anegetive
Apositive − Anegetive
Haemolysis ratio (%) =
× 100
where Asample, Anegative and Apositive are the absorbance of the super-
natant of the samples, the negative control, and positive control,
respectively.
3. Results and discussion
3.3. In vitro drug release study
3.1. Synthesis and characterization of mPEG-PCL-SS-DOX and
mPEG-PCL-SS-DTX
To evaluate the reduction-sensitive release behaviour of mPEG-
PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) mixed micelles, the in vitro
drug release studies were investigated in the presence or absence
of DTT. The release profiles of DOX and DTX are illustrated in Fig. 3a
and Fig. 3b, respectively. As expected, the in vitro DOX and DTX
release processes were reduction-responsive. As the concentra-
tion of DTT increased from 0 mM to 10 mM, the DOX release was
increased to 94.3% in 36 h, basically achieving a complete release.
For DTX, the DTX release was increased from 9.6% to 27.7% in
48 h. These results suggested that the reduction-sensitive mixed
micelles would be highly desirable for targeted cancer therapy
because it could avoid premature drug release in the blood stream.
In addition, compared with free DOX drugs (Fig. S1), the polymeric
micelles with or without DTT showed in vitro sustained release
behaviour. More importantly, as soon as the mixed micelles were
absorbed by tumour cells via endocytosis, the abundant GSH would
cleave the disulfide bonds, resulting in the targeting release of DOX
and DTX.
The typical schematic illustration for the synthesis of mPEG-
PCL-SS-DOX is depicted in Fig. 1a. The structure of the synthesized
prodrugs was characterized by 1H NMR and FTIR. In the 1H NMR
spectrum for mPEG-PCL-SS-DOX (Fig. 1b), the appearance of sig-
nals at 3.24 ppm (a) and 1.30-2.26 ppm (b, c, d) correspond to
methyl of mPEG and methylene of PCL, respectively. Signals appear-
ing at 2.76-2.96 ppm (e, f) were attributed to the ortho- and
meta-methylene of disulfide (-CH2-CH2-S-S-CH2-CH2-). Peaks at
approximately 4.00-8.00 ppm (g, h, i, j, k) belonged to the character-
istic peaks of DOX. All the facts indicate that mPEG-PCL-SS-DOX was
successfully synthesized. The 1H NMR spectrum of mPEG-PCL-SS-
DTX showed characteristic peaks of DTX at 5.40–7.62 ppm (l, m, n)
in Fig. 1c, demonstrating the successful synthesis of mPEG-PCL-SS-
DTX. The mPEG-PCL-SS-DOX was further characterized by FTIR. For
comparison, DOX, mPEG-PCL and mPEG-PCL-SS-COOH were also
characterized. The results are shown in Fig. 1d. In the FTIR spectra
of mPEG-PCL and mPEG-PCL-SS-COOH, sharp bands at 3438 cm−1
were attributed to stretching vibrations of −OH. The absorption
bands at 1725 and 1724 cm−1 were due to a stretching vibration
peak of C O. In the FTIR spectra of DOX and mPEG-PCL-SS-DOX,
the broad band in 3500–3200 cm−1 was attributed to the overlap-
ping of O-H and N-H stretching vibration. The band at 1456 cm−1
was ascribed to the N-H deformation in secondary amine structure,
originating from the linkage between the −COOH group in mPEG-
PCL-SS-COOH and −NH2 group in DOX, thus further confirming the
successful conjugation of DOX to mPEG-PCL-SS-COOH.
3.4. In vitro cellular uptake
The cellular uptake behaviour of mPEG-PCL-SS-DOX/mPEG-
PCL-SS-DTX (1:1) mixed micelles was further studied using MCF-7
cells. For comparison, the free DOX and blank control group were
also investigated. As DOX possessed the characteristic of fluores-
cence, the accumulation of mixed micelles could be in situ studied
by flow cytometry and fluorescence microscopy imaging. In gen-
eral, the cytotoxicity is directly related to the amount of drug that
accumulates in cells, which is reflected by the intensity of fluores-
cence emitted by DOX. Fig. 4a shows the cellular uptake efficiency
of the free DOX and mixed micelles compared with that of the blank
control group as determined by flow cytometry. Cells treated with
mixed micelles showed larger fluorescence peak shifts than the
cells treated with free DOX, indicating that more DOX accumulated
in cells treated with mixed micelles than in those treated with free
DOX. These results implied that the mixed micelles were more effi-
cient in delivering DOX and DTX molecules into cells, thus leading
to better treatment. Furthermore, the cellular uptake of free DOX
and mixed micelles by the MCF-7 cells were visualized by fluores-
cence microscopy imaging and their images are shown in Fig. 4b.
Consistent with the flow cytometry results, it showed that the
cells treated with the mixed micelles accumulated more DOX than
the cells treated with free drug based on the visible fluorescence
intensity from the cells. Therefore, mPEG-PCL-SS-DOX/mPEG-PCL-
SS-DTXmixed micelles can be efficientlytaken up by cells and result
in a higher concentration of drugs in cancer cells to realise effective
therapy.
3.2. Characterization of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX
(1:1) mixed micelles
The CMC of mixed micelles was determined by plotting the
fluorescence intensity ratio I373/I383 against the micelle concentra-
tion in a logarithmic scale based on our previous reported method
[29]. From Fig. 2a, the fluorescence intensity ratio decreased with
the increase of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) con-
centration due to the solubilization of pyrene molecules inside the
micellar structures. The intersection point of the two curves was
used to determine the CMC. The result showed that mixed micelles
had a low CMC of 1.8 × 10−4 mM, which was much lower than the
common surfactants of lower molecular weight [30]. In principle,
the low CMC value indicated a higher stability in vivo, guaranteeing
the micellar structural integrity before reaching the targeting site.
DLS (Fig. 2b) showed that the mixed micelles had a mean diameter
of 223.7 nm with a narrow size distribution (PDI=0.005). The TEM
image in Fig. 2c demonstrated that the as-prepared mixed micelles
had a spherical morphology with well dispersed characteristics. It
has been reported that an amphiphilic block copolymer can form a
micelle-like structure through the association of the insoluble seg-
ments with a selective solvent being a good solvent for one block
but a nonsolvent for the other [31]. In our case, one side of the
synthesized amphiphilic block copolymer was hydrophilic and the
other side was hydrophobic. Based on the molecular structure of
3.5. In vitro cytotoxicity
The in vitro antitumour efficiency of mPEG-PCL-SS-DOX/mPEG-
PCL-SS-DTX (1:1) mixed micelles was evaluated with an MTT assay
using MCF-7 cells. For comparison, the control group of free DOX,
free DTX, free DOX/DTX, mPEG-PCL-SS-DOX micelles and mPEG-
PCL-SS-DTX micelles were also studied. Our previous work showed

J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
453
Fig. 1. Typical schematic illustration of the synthesis of mPEG-PCL-SS-DOX (a), 1H-NMR spectra of mPEG-PCL-SS-DOX in DMSO-d6 (b) and mPEG-PCL-SS-DTX in CDCl3 (c),
FTIR spectra of DOX, mPEG-PCL, mPEG-PCL-SS-COOH and mPEG-PCL-SS-DOX (d).
that mPEG-PCL-SS-COOH had good biocompatibility and did not
exhibit significant cytotoxicity to MCF-7 cells [24]. Here, MCF-7
cells were treated with various concentrations of DOX plus DTX
from 1 nM to 10000 nM. The cell inhibition ratio of the mixed
micelles and pure DOX/DTX free drugs are presented in Fig. 5, while
the corresponding IC50 values are listed in Table 1. Based on the
cell inhibition ratio, the cytotoxicity of mixed micelles was nearly
equivalent with the mixture of DOX and DTX at the same dosages.
However, the cytotoxicity of single mPEG-PCL-SS-DOX micelles
and mPEG-PCL-SS-DTX micelles were lower than single free DOX

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J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
Fig. 2. The fluorescence ratio (I373/I383) of pyrene at different concentrations of mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) (a) (mean SD, n = 3), the size distribution
determined by DLS (b) and TEM image result (c).
Fig. 3. In vitro release profiles of DOX and DTX from mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) mixed micelles in the absence and presence of DTT. DOX release profile (a),
DTX release profile (b) (mean SD, n = 3).
Table 1
time for 24 h is insufficient to make the disulfide bond in high con-
tact with intracellular GSH for the sufficient release of DOX and
DTX from mixed micelles. The mixed micelles exhibited a lower
IC50 values of free drugs DOX/DTX (1:1) and mPEG-PCL-SS-DOX/mPEG-PCL-SS-
DTX (1:1) mixed micelles against MCF-7 cells after 24 h, 48 h and 72 h incubations
(mean SD, n = 3).
IC50 than the mixture of DOX and DTX after 48 h and 72 h incuba-
Treatment
IC50 (nM)
24 h
tion. For example, the IC50 of mixed micelles was 12.0 nM while
that of the mixture of DOX and DTX was 27.8 nM for 48 h incu-
bation. More importantly, after 72 h incubation, the IC50 of mixed
micelles (2.5 nM) was roughly one-tenth of the value of the mixture
of DOX and DTX (26.2 nM). The results suggested that the mixed
micelles had an excellent sustained drug release behaviour, which
could promote the effectiveness of the cancer therapy. Although the
MTT assay showed that the mixed micelles and DOX/DTX free drugs
had almost equivalent cytotoxicity, the mixed micelles had many
advantages over the mixture of drugs. For example, the mixture of
pure drugs often causes systemic toxicity to the patient whereas the
mixed micelles reduced the side effects of chemotherapy. Further-
48 h
72 h
DOX/DTX (1:1)
111.1 2.7 27.8 0.9 26.2 2.6
mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) 465.2 1.4 12.0 2.8 2.5 1.6
and DTX, respectively, as shown in Fig. S2. Cytotoxicity studies
demonstrated that compared with single drug micelles, the mixed
micelles consisting of two chemotherapeutic drugs exhibited supe-
rior anti-tumour efficacy, suggesting the existence of synergistic
effects among the two drugs. For the IC50 after 24 h incubation,
mixed micelles exhibited a higher IC50 (465.2 nM) than the mix-
ture of DOX and DTX (111.1 nM). This indicates that the incubation

J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
455
Fig. 4. Flow cytometry results (a) and fluorescence microscopy images (b) of MCF-7 cells after incubation with blank, free DOX, mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1)
mixed micelles for 4 h.
Fig. 5. The cell inhibition ratio of free drugs DOX/DTX (1:1) and mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX (1:1) mixed micelles to MCF-7 cells after 24 h, 48 h and 72 h incubations
(mean SD, n = 3).

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J. Wu et al. / Colloids and Surfaces B: Biointerfaces 161 (2018) 449–456
Acknowledgements
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (NSFC, No. 21373126 and
No.21673128).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.colsurfb.2017.11.
011.
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In summary, we proposed a new reduction-sensitive mixed
micellar system assembled from amphiphilic prodrugs (mPEG-PCL-
SS-DOX and mPEG-PCL-SS-DTX) for self-codelivery of DOX and
DTX. The mixed micelles with good stability showed the triggered
release of both DOX and DTX in the presence of DTT, mimicking the
intracellular reductive environment. Importantly, compared with
pure drug mixtures, the reduction-sensitive mixed micelles were
capable of a higher cellular uptake and almost similar cytotoxic-
ity. The mixed micelles had many advantages over the pure drug
mixtures such as long-term circulation in the blood, controlled
drug release, minimised drug distribution to normal organs and
lower toxicity. Moreover, the mixed micelles exhibited a lower IC50
than the pure drug mixtures after 48 h and 72 h incubations, which
offered the promise of enhanced efficacy and dose-reduction. The
present proposed reduction-sensitive mixed micelles assembled
from amphiphilic prodrugs for self-codelivery of the active drugs
might open the door for selective codelivery of drugs to the intra-
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