European Journal of Pharmaceutics and Biopharmaceutics 127 (2018) 177–182

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European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Research paper

Hierarchical drug release of pH-sensitive liposomes encapsulating aqueous T

two phase system
Xunan Zhanga, Wei Zonga, Hongmei Bib, Kunming Zhaoc, Thomas Fuhsa, Ying Huc,
⁎
Wenlong Chengd, Xiaojun Hana,
a
State Key Laboratory of Urban Water Resource and Environment, School of Chemical Engineering and Technology, Harbin Institute of Technology, 92 West Da-Zhi Street,
China
b
College of Science, Heilongjiang Bayi Agricultural University, Daqing 163319, China
c
School of Life Science and Technology, Harbin Institute of Technology. Harbin Institute of Technology, 92 West Da-Zhi Street, China
d
Department of Chemical Engineering, Monash University, Victoria 3800, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: As promising drug delivery vehicles, previous investigations of liposomes as carriers are primarily focused on
Liposome insertion and modification of lipid membrane interfaces. The utility of the inner core seems to be overlooked.
Aqueous two phase system (ATPS) Herein, we developed pH-sensitive liposomes (PSLs) containing an aqueous two phase system (ATPS), and in-
Hierarchical drug release triguingly discovered their hierarchical release under acidic stimuli. ATPS containing two polymers (poly
(ethylene glycol) (PEG) and dextran) is homogeneous above phase transition temperature when producing
ATPS-liposomes, and separated into PEG-rich phase and dextran-rich phase after cooling down to room tem-
perature. The overall release time of ATPS-liposomes is divided into two stages and prolonged compared to
simple aqueous liposomes. The unique release profile is due to the disproportional distribution of drugs in two
phases. Doxorubicin (DOX) is loaded in the ATPS-liposomes, and their half maximum inhibition concentration
on HeLa cells is 0.018 μmol L−1, which means 27.5 fold increase in inhibition efficiency over free DOX.

1. Introduction
Liposomes have attracted considerable attention [1–5] as promising
drug delivery systems in the field of medicine. Liposomes can carry
various types of drugs, for instance, hydrophilic drugs in the aqueous
core, lipophilic drugs in the lipid bilayer, and amphiphilic drugs par-
titioned at the surface of the bilayers [6]. Cancerous tissues usually
exhibit imparities in their micro-environment as compared to normal
tissues [7–9], which forms the foundation of pH-sensitive liposomes
(PSLs) [10–13].
PSLs are designed to be stable at pH of blood (7.4) and degrade at
lower pH (≤6.0) presenting in tumor surroundings [14] or sites of
inflammation [15]. One example of PSL contains fusogenic peptides on
the membrane surface [16]. The fusogenic peptides are also known as
low pH insertion peptides, which are unimolecular and soluble in water
at neutral pH conditions, whereas turn into a helix structural HII phase
inserting into the membrane to fuse the liposome with the cell mem-
brane at acidic pH conditions. Therefore, in areas of low pH, the peptide
will enhance the cellular uptake of liposomes and the release of cargo
[17–19]. Another approach to design PSLs is using pH sensitive
functional groups between the poly(ethylene glycol) (PEG) and lipid
bilayers. The hydrolysis of the functional groups such as ester or hy-
drazine at low pH releases PEG, and consequently enables the lipo-
somes available for local drug delivery [20]. The principle of the sys-
tems is that conjugated compounds provide targeting, as well as
shielding the liposomes from plasma proteins, and finally release the
drug at the tumor sites [21]. Both type of PSLs exhibit better drug re-
lease behaviors as compared to the unmodified liposomes [22]. Cur-
rently in this field, most of scientists focus on the membranes and
grafting moieties to increase the efficiency of drug release of PSLs.
However, the role of the inner core of PSLs on drug release seems to be
neglected.
Herein, we introduced aquous two phase system (ATPS) composed
of PEG and dextran into PSLs. Compared to the exponential drug release
of common PSLs, the novel ATPS-PSLs possess a unique hierarchical
release profile. ATPS solutions with precise proportion will separate
into two phases below transition temperature and merge into one single
phase beyond transition temperature. The separation caused by mac-
romolecular crowding leads [23] to disproportional distribution of so-
lute (such as DOX) in each phase, hence the hierarchical DOX release

⁎
Corresponding author.
E-mail address: hanxiaojun@hit.edu.cn (X. Han).

https://doi.org/10.1016/j.ejpb.2018.02.021
Received 23 August 2017; Received in revised form 26 December 2017; Accepted 16 February 2018
Available online 17 February 2018
0939-6411/ © 2018 Elsevier B.V. All rights reserved.
X. Zhang et al. European Journal of Pharmaceutics and Biopharmaceutics 127 (2018) 177–182

Fig. 1. (a) Phase diagrams of ATPS at 65 °C and

37 °C (inset). (b) Initial mix-up of ATPS, (c) he-
ated up at 65 °C and (d) cooled down under
bright field and corresponding status (e–g) under
green fluorescence light. (h) Optical microscope
image of ATPS-GUVs at room temperature. ATPS-
GUV under (i) transmitted DIC, (j) red fluores-
cence with TR-PE labelled DPPC/Chol, (k) green
filter with FITC labelled dextran and (l) their
merged image. The scale bars are (h) 20 μm and
(i–l) 10 μm.

from ATPS-PSLs can be realized. Furthermore, the new system exhibits observation of ATPS separation. The following experiments were car-
better inhibition efficiency than common PSLs, because of longer re- ried out by ATPS-large unilamellar vesicles (LUVs) (∼100 nm in dia-
lease time provided by hierarchical release, and higher uptake rate of meter), which is in the size range for enhanced permeability and re-
doxorubicin (DOX). The new system is considerably stable. From all tention (EPR) effect. It seemed plausible to assume that the phase
above, the ATPS-PSLs system is quite competent as drug delivery separation in LUVs was the same as in GUVs.
system. Ideally drug loaded PSLs should remain stable in normal body fluid,
but destabilized at acidic conditions to target cancer cells without
premature dumping of the cargo in the blood [26]. Recent studies have
2. Results and discussion
been carried out using various lipids, or mixtures of cationic and pH-
sensitive lipids to prepare drug loaded liposomes [13,27,28]. The in-
Neutral polymer solutions mixed at certain concentration can lead
fluence of the inner core on drug release is often neglected. In the
to the formation of distinct thermodynamic phases [24], driven by
following experiments, DOX loaded ATPS-LUVs were prepared and
predominantly repulsive interactions between polymers. Fig. 1a
used for controlled release. Firstly, LUVs were prepared at 65 °C by
showed the phase diagrams for ATPS at 65 °C and 37 °C, determined by
extrusion. At this temperature the PEG and dextran were homo-
the “cloud point determination” method [25]. The curves in Fig. 1a
geneously mixed, as shown in Scheme 1a. After cooling down to room
show the critical status of the solution at different temperatures. The
temperature, the solution inside LUVs became aqueous two phase of
areas below the curve stand for the homogeneous phase of the two
dextran-rich phase as core and PEG-rich phase layer surrounding it, as
components, while the areas above the curves mean heterogeneous
shown in Scheme 1b. At this stage, DOX were distributed dis-
phases at each temperature. The ATPS-liposomes were prepared as the
proportionally in these two phases. Changing to acidic condition, the
ATPS in homogeneous phase at 65 °C, and further experiments were
release of DOX happened starting from the outer PEG-rich layer, fol-
carried out as the ATPS in heterogeneous phase at 37 °C. In our case, we
lowed by inner dextran-rich phase as shown in Scheme 1 c and d.
chose a solution containing 3.00 wt% PEG and 3.35 wt% dextran.
Passive targeting of drug loaded nano-vesicles relying on the en-
Fig. 1b and e illustrate that the initial status of ATPS at 65 °C, turned
hanced permeability and retention (EPR) effect [29] can overcome the
into a single phase above 65 °C (Fig. 1c and f), and became two separate
disadvantages exhibited by traditional drugs, including poor solubility,
phases again below 65 °C (Fig. 1d and g). From Fig. 1g, FITC labelled
limited stability, and, in particular, lack of selectivity. Those drawbacks
Dextran was the lower strata in the bulk solution, which clearly shows
of traditional drugs result in nonspecific toxicity to normal cells and
the two phase separation. In the giant unilamellar vesicles (GUVs), the
dose escalation necessary to annihilate tumor cells [30,31]. The DOX
aqueous core also separated into two phases as shown in Fig. 1h and i.
loaded ATPS-LUVs possess the passive targeting property. The release
Since Llipid bilayer membrane was labelled by TR-PE, the red circle
profile of DOX from ATPS-LUV exhibited a unique character compared
means a GUV (Fig. 1j). Using FITC labelled dextran, the fluorescense
with that from LUVs at lower pH solution (pH = 5). Unlike the ex-
image of the same GUV was obtained (Fig. 1k). After merging Fig. 1j
ponential release tendency (Fig. 2a) of DOX from DOX-LUV [13], the
and k, it is clearly seen that dextran-rich phase was in the inner layer
DOX release profile from ATPS-LUV presents two distinct stages as
(Fig. 1l). On the large scale preparations in bottles, dextran-rich phase
shown in Fig. 2b. The first phase completed at 8 h, the release per-
forms at the bottom due to their higher density. On micro-scale, the
centage is 48.54%. Meanwhile, the corresponding release percentage of
contribution of gravity is negligible. PEG-rich phase with higher affinity
LUVs is 63.98%. And the release percentage of ATPS-LUV at 11 h is
with the lipid bilayer surface was expected to wet the bilayer pre-
62.84%. Furthermore, the release of LUV stops at 17 h, and the release
ferentially. The abovementioned GUV data were only for microscopic

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X. Zhang et al. European Journal of Pharmaceutics and Biopharmaceutics 127 (2018) 177–182

Scheme 1. Schematic illustration of preparation and stimulation of ATPS-liposomes. (a) Liposomes produced in the DOX loaded ATPS above transition temperature. (b) Inner solution
separation to be two phases with disproportional DOX concentrations in each phase. (c) DOX release from PEG-rich phase triggered in the acidic conditions. (d) DOX release from dextran-
rich phase.

percentage is 84.87%. The second release stage of ATPS-LUV finishes
around 23 h and the total release percentage is 85.85%. They reach the
similar terrace due to the same initial DOX concentration which is the
predominant driven force of simple diffusion [32]. We presumed that
the reason of this hierarchical release profile is due to the dispropor-
tionate DOX distribution in ATPS, and the experiments verified our
assumption (SI section 1). The DOX concentrations in PEG-rich phase
and dextran-rich phase were 8.3 ± 0.4 μg mL−1 and
6.2 ± 0.6 μg mL−1 respectively determined by fluorescence spectro-
scopy, while the average concentration was 7.5 μg mL−1. Due to the
intriguing mechanism of ATPS-LUV, the release time of ATPS-LUV was
prolonged 6 h more than that of LUVs. The existence of two phase re-
lease behavior indicates buffered effect of ATPS, which can slow down
the drug release to obtain the longer drug effect in the body.
A series of carriers were stored at 4 °C after producing for further
experiments. Stabilities of various carriers were evaluated by dynamic
light scattering (DLS), polymer dispersity index (PDI) and ζ-potential
(SI section 2). The diameters of LUVs and ATPS-LUVs were about
160 nm and 100 nm, respectively, no matter whether DOX was loaded
or not. The diameter and PDI of each carrier remained almost un-
changed over 30 days, although ATPS caused a small amount increase
in PDI. The changes of ζ-potentials do not exhibit specific patterns, but
the values did not go down below −25 mV, which indicates the lipo-
somes did not coagulate over a month.
The composition of ATPS-LUV was DPPC and Chol (80:20 mol%),
which are the constituents of cell membranes [33,34]. Average size of
ATPS-LUVs was about 100 nm (Fig. S3) that fit in the optimum range
for tumor delivery in accordance to ERP effect. Inhibition of free DOX,
DOX loaded LUV and DOX loaded ATPS-LUV on HeLa cells after 12, 24,
36, 48 h were summarized in Fig. 3 estimated by MTT assay [35,36]. As
expected, the increase of DOX concentration in each group resulted in a
decreased population of HeLa cells, and the necessary concentration for
decreasing the cell viability was reduced with increasing incubation
time. In addition, DOX loaded LUV showed a higher cytotoxicity on
HeLa cells than free DOX solution at each concentration [37]. Intrigu-
ingly, the performance of DOX loaded ATPS-LUVs were even better,
meanwhile they showed insignificant toxicity on normal cells (SI sec-
tion 3). The half maximal inhibitory concentrations (IC50) of DOX so-
lution, DOX loaded LUV, DOX loaded ATPS-LUV were 0.495 μmol L−1
and 0.119 μmol L−1 and 0.018 μmol L−1 calculated by IBM SPSS Sta-
tistics respectively. These results demonstrated the outstanding per-
formance of ATPS-LUV as drug delivery system.
In order to confirm the carrier and drug distribution inside HeLa
cells, NBD-PE labelled DOX-ATPS-LUVs were incubated together with
HeLa cells for 12 h. Fig. 4a presented the nuclei of HeLa cells stained
with DAPI. DAPI stains the nuclei compartment of both live and dead
cells. Fig. 4b and c were NBD-PE and DOX distribution inside HeLa cells
respectively. Fig. 4d was the merged image of Fig. 4a, b and c. From
Fig. 4b, it was clearly seen that ATPS-LUVs were taken up by the tumor
cells and located in their cytoplasm (green parts in Fig. 4b). The image
in Fig. 4c indicated that the DOX are released mostly inside the cells
with scarce premature release in the exterior of the HeLa cells.
The LUVs can promote the uptake of DOX [38], and ATPS-LUVs can
further escalate the uptake of DOX, as shown in Fig. 5a. The possible
reason of the escalation in cytotoxicity of DOX-ATPS-LUVs is due to
their slower release profile. Although the localization of the carriers are
similar (Fig. b, c, and d), the uptake of DOX-ATPS-LUVs was better than
that of DOX-LUVs. DOX, DOX-LUVs and DOX-ATPS-LUVs of equivalent
concentration 10 μM were incubated with HeLa cells for 5 h (SI section
4), separately, and determined the uptake of each group by their mean

100
a 100 b
Comulative release (%)

Comulative release (%)

80
pH=5.0 80
60 pH=5.0
60
40 40
20 20

0 pH=7.4 0 pH=7.4
0 5 10 15 20 0 5 10 15 20 25
Time (h) Time (h)
Fig. 2. DOX release profiles of (a) LUVs, and (b) ATPS-LUVs. (n = 3, error bar = standard deviation).

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X. Zhang et al. European Journal of Pharmaceutics and Biopharmaceutics 127 (2018) 177–182

12 h 24 h
1.0 1.0

0.8 0.8

Cell viability
Cell viability
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 0.1 0.25 0.5 1 5 10 0 0.1 0.25 0.5 1 5 10
Concentration of DOX (ȝM) Concentration of DOX (ȝM)

36 h 48 h
1.0 1.0

0.8 0.8

Cell viability
Cell viability

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 0.1 0.25 0.5 1 5 10 0 0.1 0.25 0.5 1 5 10

Concentration of DOX (ȝM) Concentration of DOX (ȝM)

Control group : Negative control LUV ATPS-LUV

Experimental group : Positive control DOX DOX-LUV DOX-ATPS-LUV

Fig. 3. The viability of HeLa cells with treatments of control group and experimental group after 12 h, 24 h, 36 h and 48 h (n = 3, error bar = standard deviation).

a 600 b
500
MCF (a.u.)

400
300
200
100
0
DOX DOX-LUVs DOX-ATPS-LUVs

c d

Fig. 5. (a) Mean cellular fluorescent determination of uptake of DOX, DOX-LUVs, and
DOX-ATPS-LUVs by HeLa cells for 5 h. (b–d) Representative fluorescence microscopic
images of DOX, DOX-LUVs, and DOX-ATPS-LUVs uptake by HeLa cells, respectively. Each
test collects 10,000 events and repeats 3 times. The scale bar is 40 μm.

Fig. 4. Fluorescence images of the uptake of NBD-PE labeled DOX loaded

ATPS–liposomes by HeLa cells. (a) DAPI channel, (b) NBD-PE channel, (c) DOX channel,
(d) merged image of (a), (b) and (c). The scale bar is 20 μm.
cellular fluorescence (MCF) using a flow cytometry. The MCF of each
group was 265.79 ± 18.74, 370.88 ± 28.56 and 510.17 ± 24.31.
These results explained the best inhibitory efficiency of ATPS-LUVs.
3. Conclusion
In summary, two different neutral polymers with a delicate pro-
portion can form a unique aqueous system, which leads to the forma-
tion of distinct phases, driven by repulsive interactions between poly-
mers, and turns into a homogeneous phase above critical temperature.
Soluble drugs such as DOX is distributed disproportionally in ATPS,

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X. Zhang et al.
which results in hierarchical drug release. We successfully prepared
ATPS and encapsulated it in liposomes. ATPS-LUVs exhibited a few
merits as compared to free DOX and simple LUVs, such as longer sus-
tained release time, higher uptake efficiency, meanwhile higher in-
hibition efficiency and controllable composition of the inner core,
which show great potential in pharmacologic therapy of cancer treat-
ment.
4. Experimental
4.1. Materials and characterizations
Poly(ethylene glycol) (PEG) (20 kDa), dextran (500 kDa), doxor-
ubicin (DOX) , poly-L-lysine hydrobromide (PLL) (40–60 kDa) and
fluorescein isothiocyanate–dextran (FITC-dextran, 200 kDa) were pur-
chased from Sigma (China). 1,2-dihexadecanoyl-sn-glycero-3-pho-
phocholine (DPPC) and cholesterol (Chol) were purchased from Avanti
Polar Lipids (USA). 1,2-dioleoyl-sn- glycero -3-phosphoeth-anolamine-
N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and dihexadecanol-sn-
glycero-3-Phosphethanolamine, triethylammonium salt (TR-PE) were
obtained from Molecular Probes (Eugene, Oregon, US). Fetal bovine
serum (FBS) was purchased from Thermo Fisher Scientific (China).
HeLa cells and human umbilical vein endothelial cells (HUVECs) were
purchased from American type culture collection (ATCC). 4-(2- hy-
droxyethyl) piperazine-1-ethane-sulfonic acid (HEPES) and 4′,6-diami-
dino-2-phenylindole (DAPI) were purchased from Biotopped (China).
Propidium iodide (PI) was obtained from Beyotime (China). Absolute
ethanol was purchased from FuYu Chemicals (China). 3-(4,5-D- -im-
ethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and di-
methylsulfoxide (DMSO) were purchased from Sigma (China). Dialysis
bag (8–14 kDa) was obtained from Solarbio, (China). Millipore Milli-Q
water with a resistivity of 18.2 MΩ cm was used for solution prepara-
tion.
Microscopy images were taken with a fluorescence microscope
(Nikon 80i, Japan). Differential interference contrast (DIC) images were
taken with a Leica DMi8 microscope. Mini-Extruder Kit was purchased
from Avanti Polar Lipids. Drug release profiles were acquired on a
Fluorescence spectrometer (Perkin Elmer LS55). The cells were grown
and maintained in RPMI-1640 (GIBCO). MTT experiments were mea-
sured using a microplate reader (Infinite M200, Tecan).
4.2. Preparation of DOX-ATPS-GUV
ITO-coated glass coverslips were cleaned in ethanol and water each
for 15 min by sonication and then dried by N2. DPPC/Chol were dis-
solved in chloroform with a concentration of 5 mg mL−1 as stocking
solution. ATPS was prepared by dissolving 3 wt% PEG (20 kDa) and
3.35 wt% dextran (500 kDa) in deionized (DI) water, and DOX was
added to make a final concentration of 10 μM. The lipid solution was
dropped onto ITO electrode surfaces, followed by drying under vacuum
for 2 h to form thin films. The ITOs were separated by a rectangular
polytetrafluoroethylene (PTFE) spacer filled with DOX-ATPS solution.
Subsequently, an AC-electric field (2.5 V mm−1, 10 Hz) was applied for
4 h to generate giant unilamellar vesicles GUVs [39]. In order to purify
GUVs, the suspension was dialyzed by dialysis bag in DI water for 24 h
before carrying out other experiments. The AC-electric field was ap-
plied to form GUVs at 65 °C. GUVs were only produced for observing
the aqueous two phase inside liposomes due to the resolution limitation
of the microscope. The following experiments were carried out using
LUVs. Their diameter was more appropriate for in vitro and in vivo
experiments [25].
4.3. Preparation of DOX-ATPS-LUV
DOX-ATPS-LUVs were produced by mini-extruder. The lipid
stocking solution was dried in a test tube and then dissolved in DOX
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European Journal of Pharmaceutics and Biopharmaceutics 127 (2018) 177–182
loaded ATPS, followed by vortexing for 3 min, preheating at 65 °C. The
solution then went through the polycarbonate membranes with pores of
200 nm diameter for 11 times back and forth with syringes. The ob-
tained LUVs are about 160 nm. The LUV suspension was also purified
with the same method as GUVs.
4.4. Observation of ATPS-GUV
Coverslips were cleaned using a plasma cleaner (Zepto/Atto, USA)
for 30 min, then coated with 1 mg mL−1 PLL. After washing with
100 nM NaCl solution, the coated coverslips were immersed into GUV
solution for at least 15 min, and followed by washing with NaCl solu-
tion again before observation.
4.5. Estimation of stability
Stability of LUVs, ATPS-LUVs and DOX loaded LUVs and ATPS-LUVs
were evaluated by dynamic light scattering (DLS), polymer dispersity
index (PDI) and ζ-potential. DLS was tested using a material refringence
of 1.590 and a dispersant index of 1.33.
4.6. In vitro controlled release of DOX-ATPS-LUV
The in vitro release of DOX-ATPS-LUVs was performed by a dialysis
method. The DOX-ATPS-LUV suspension (0.5 mL) was added into a
dialysis membrane (molecular weight cut-off 8–14 kDa, MD 44,
Solarbio, China) that was subsequently placed into 20 mL buffer solu-
tion (sodium hydrogen phosphate-citric acid, pH = 5 and pH = 7.4
respectively) under magnetic stirring at 37 °C. At desirable time inter-
vals, 2 mL of supernatant were taken to measure fluorescent intensity
and then returned back to mother solution. The release percentage at
each time interval was obtained by the amount of DOX released at that
time over the total amount of DOX inside the LUVs.
4.7. Cell culture and viability
The cells were grown in a RPMI-1640 (GIBCO) medium with 10%
(v/v) fetal bovine serum (FBS). The cells were maintained in an in-
cubator at 37 °C in an atmosphere of 5% CO2. The cells were seeded in
96-well plates with a density of 6000 viable cells per well and incubated
for 24 h for cell attachment. The antitumor activity of the compounds
was tested by standard MTT experiments. Various concentration of DOX
solutions were prepared. After 24 h, 20 µL MTT solution at a con-
centration of 5 mg mL−1 dissolved in phosphate buffer solution (PBS,
pH 7.4) was added to each well. After another 4 h incubation, the
medium was removed and 100 µL dimethylsulfoxide (DMSO) was
added into each well. The intensity of the absorbance was measured
using an automatic microplate reader at a wavelength of 495 nm. The
results were expressed as mean values ± standard deviation of 3
measurements.
4.8. Uptake of DOX-ATPS-LUVs by HeLa cells and their localization in cells
To evaluate the uptake of DOX-ATPS-LUVs by HeLa cells, lipid was
labelled with NBD-PE. Cells were seeded in a 24-well plate in RPMI-
1640 medium overnight. Fluorescence-labelled liposomes were sus-
pended over a bath sonicator for 3 min, and 0.5 mL solution was added
to the cultured cells. After a certain period of time, the cells were fixed
with 4% paraformaldehyde for 10 min and washed three times with
PBS, then stained with DAPI (100 ng mL−1) for 3 min. After washing
with PBS, the coverslips were mounted immediately and cells were
directly visualized by a fluorescence microscope.
Conflicts of interest
The authors declare no competing financial interest.
X. Zhang et al.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant nos. 21773050, 21650110452, 21528501,
21503072), and China Postdoctoral Science Foundation
(201620162016).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.ejpb.2018.02.021.
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