Photo-cross-linked poly(ether amine) micelles for controlled drug release

Article abstract of DOI:10.1039/c5ra22679a

In order to improve the stability of micelles and decrease the burst release of loaded drugs, photo-cross-linked micelles were prepared via photodimerization of the coumarin moiety on amphiphilic poly(ether amine) (PEAC). The structures of the obtained monomer and polymers were confirmed by Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), ¹H NMR and ¹³C NMR (Nuclear Magnetic Resonance, NMR). PEAC could self-assemble into micelles by directly dispersing in water with a hydrophobic coumarin core and a hydrophilic poly(ethylene glycol) (PEG) shell. The photo-cross-linking process of the PEAC micelles was monitored by UV-vis spectroscopy. The morphology and size distribution of the micelles was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Anticancer drug doxorubicin (DOX) was loaded into the micelles during the process of micelle formation. Photo-cross-linked micelles showed slower drug release and cellular uptake in comparison with the uncross-linked micelles. And both DOX-loaded micelles displayed pH-sensitive release behaviours. Moreover, the DOX-loaded photo-cross-linked micelles exhibit comparative anticancer efficacy as free DOX. These results indicated that photo-cross-linked PEAC micelles can be used as potential drug carriers for intelligent drug delivery.

Full text of DOI:10.1039/c5ra22679a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Photo-cross-linked poly(ether amine) micelles for
controlled drug release†
Cite this: RSC Adv., 2015, 5, 105880
a
a
a
a
a
a
Haozhe He, Yanrong Ren,* Yuge Dou, Tao Ding, Xiaomin Fang, Yuanqing Xu,
a
a
b
Hao Xu, Wenkai Zhang and Zhigang Xie*
In order to improve the stability of micelles and decrease the burst release of loaded drugs, photo-cross-
linked micelles were prepared via photodimerization of the coumarin moiety on amphiphilic poly(ether
amine) (PEAC). The structures of the obtained monomer and polymers were confirmed by Fourier
1
13
transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), H NMR and C NMR
Nuclear Magnetic Resonance, NMR). PEAC could self-assemble into micelles by directly dispersing in
(
water with a hydrophobic coumarin core and a hydrophilic poly(ethylene glycol) (PEG) shell. The photo-
cross-linking process of the PEAC micelles was monitored by UV-vis spectroscopy. The morphology and
size distribution of the micelles was characterized by transmission electron microscopy (TEM) and
dynamic light scattering (DLS). Anticancer drug doxorubicin (DOX) was loaded into the micelles during
the process of micelle formation. Photo-cross-linked micelles showed slower drug release and cellular
uptake in comparison with the uncross-linked micelles. And both DOX-loaded micelles displayed pH-
sensitive release behaviours. Moreover, the DOX-loaded photo-cross-linked micelles exhibit comparative
anticancer efficacy as free DOX. These results indicated that photo-cross-linked PEAC micelles can be
used as potential drug carriers for intelligent drug delivery.
Received 29th October 2015
Accepted 30th November 2015
DOI: 10.1039/c5ra22679a
www.rsc.org/advances
entrapped drugs in blood stream nor release drugs specically
in the interior of targeted cells in a controlled manner.
In order to improve the stability of micelles and decrease the
Introduction
1,2,15–18
Polymeric nanocarriers have been extensively investigated and
used in drug delivery. In order to obtain high efficacy of deliv- burst release of loaded drugs, chemical and physical cross-
ered drugs and enhanced therapeutic effect, a nanocarrier linking have been recognized as the powerful approaches and
1
9,20
should meet the following requirements: (i) maintaining high widely used in previous work.
structural stability in blood aer intravenous injection and cross-linking, photo-initiated cross-linking
eliminating undesirable drug release before reaching the target considerable interest due to rapid, effective, well-controlled
Comparing with other ways of
21–23
has attracted
21–28
site, (ii) releasing drugs specically and completely within tar- unique advantages in practical applications.
Among
geted cells, (iii) as a nanocarrier itself being non-toxic, and not various photo-crosslinkable molecules, the coumarin analogues
1–6
accumulate in the body. Among many potent nanocarriers, have been utilized in medicine, biology, and material
2
2,29–31
polymer micelles assembled from amphiphilic copolymers have sciences.
attracted great attention in the past few years and have been one of coumarin has also been explored for designing photo-
More recently, the reversible photodimerization
7–12
29–34
of the important candidates for intracellular drug delivery.
controllable micelles.
Coumarin has the potential to pho-
However, the micelles oen disintegrate aer dilution in uid todimerize via a photo-induced [2 + 2] cycloaddition under
blood vessel upon intravenous administration and the nano- irradiation of UV light at l > 310 nm, and the formed dimer can
carriers generally have issues of premature burst drug release be reversibly dissociate into two coumarin molecules under
1
3–17
22,29–32
within the rst several hours.
If so, micelles can neither hold irradiation of UV light at l < 260 nm.
For example, Luo and
prepared coumarin-containing telodendrimers
micelles cross-linked at 310 nm, which showed superior drug
31
coworkers
22
loading efficiency, capacity and stability. Ji and coworkers
a
College of Chemistry and Chemical Engineering, Henan Universi, Kaifeng, 475004,
P. R. China. E-mail: renyr@henu.edu.cn; Fax: +86-371-23881586; Tel: +86-371-
prepared a novel coumarin-based pH-responsive polymer,
indicating cross-linked micelles could avoid the unfavourable
premature release of loaded drugs during circulation.
However, toxic organic solvents or catalysts are oen used
during the process of synthesizing polymers. Avoiding toxic
reagents, or using low toxicity and non-toxic solvents is
2
3881586
b
State Key Laboratory of Polymer Physics and Chemistry, Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China. E-mail:
xiez@ciac.ac.cn; Fax: +86-431-85262775; Tel: +86-431-85262775
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra22679a
1
105880 | RSC Adv., 2015, 5, 105880–105888
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
2
2,31,32,35
2
important for ideal drug release systems.
In addition,
y ¼ 0.0169x + 0.0185, R ¼ 0.9992
(2)
(3)
polymeric micelles are usually prepared by rst dissolving the
copolymers in organic solvents for both blocks and following by
Herein, we made the amphiphilic
copolymers by nucleophilic addition and obtained their
micelles by directly dispersing of PEAC in aqueous solution. It is
worthy to mention that Yin and coworkers
ported the synthesis of coumarin-containing poly(ether amine) Synthesis of 5,7-dihydroxy-4-methyl coumarin (DHMC)
PEAC) and investigated its temperature-responsive behaviour.
2
y ¼ 0.0185x + 0.0175, R ¼ 0.9993
21,22
the addition of water.
here y is the absorption intensity of DOX, x is the concentration
of DOX, and R is the correlation coefficient.
36,37
previously re-
(
The mixture of phloroglucinol (0.1 mol), ethyl diacetoacetate
In this work, we further modied the synthetic method, inves-
tigated the pH-responsive and photo-crossing behaviour of
PEAC, and evaluated the potential of PEAC as intelligent drug
carriers.
(
0.12 mol) and sodium bisulfate monohydrate (3 g) were reacted
ꢁ
at 110 C for 40 min. The resulting mixture was dissolved by
anhydrous ethanol under reuxing. Then the solution was

ltered to remove the catalyst. The ltrate was poured into ice-
cold water to yield a light yellow precipitate. The product was
1
dried in vacuum to get DHMC with a yield about 94%. H NMR
Experimental details
Materials
(
1
400 MHz, DMSO): d 6.24 (t, J ¼ 9.9 Hz, 1H), 6.16 (d, J ¼ 2.2 Hz,
ꢀ1
H), 5.84 (s, 1H), 2.49 (d, J ¼ 6.6 Hz, 2H); FTIR (neat, cm
)
Phloroglucinol, sodium bisulfate monohydrate and piperazine 3200, 1732, 1616, 1110.
Pip) were purchased from Aladdin Industrial Inc., Shanghai,
(
China. Ethyl diacetoacetate, epichlorohydrin, methylbenzene,
triethylamine (TEA) were purchased from China National
Pharmaceutical Group Corporation, Beijing, China. Doxoru-
bicin (DOX) in the form of a hydrochloride salt was purchased
from Hisun Pharmaceutical Co., Ltd. Zhejiang, China.
Synthesis of 4-methyl-5,7-bi(2,3-epoxypropoxy) coumarin
DEMC)
(
The mixture of DHMC (0.01 mol), epichlorohydrin (0.1 mol) and
potassium carbonate (0.0125 mol) were dissolved in toluene
ꢁ
and reacted at 75 C for 8 hours. Then, sodium hydroxide was
Poly(ethylene glycol) diglycidyl ether (PEGDGE, M
n
¼ 500 g
mol ), 2-(4-amidinophenyl)-6-indolecarbamidine dihydro-
chloride (DAPI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
added in and reacted for another 6 hours. The resulting mixture
was dissolved by anhydrous ethanol under reuxing. Then the
solution was ltered and poured into ice-cold water. The
resulting precipitate was collected by ltration and washed with
ꢀ
1
diphenyltetrazolium bromide (MTT) were purchased from
Sigma-Aldrich Co, Shanghai, China. Lyso-Tracker Red was
purchased from Beyotime Biotechnology, China.
1
water to get DEMC with a yield about 70%. H NMR (400 MHz,
DMSO): d 6.61 (s, 1H), 6.55 (s, 1H), 6.04 (s, 1H), 4.46 (d, J ¼ 11.4
Hz, 2H), 3.98–3.85 (m, 2H), 3.39 (d, J ¼ 29.6 Hz, 4H), 2.87 (dd,
ꢀ
1
Measurements
J ¼ 10.0, 5.1 Hz, 2H), 2.76–2.66 (m, 2H); FTIR (neat, cm ) 1732,
1
1616, 1110.
H NMR spectra were characterized on a Bruker AVANCE 400 M
ꢁ
spectrometer in CDCl
3
and DMSO-d
6
at 25 C. Chemical shis
were given in parts per million with respect to tetramethylsilane Synthesis of PEAC
(
(
TMS) as an internal reference. Gel permeation chromatography
GPC) measurements were conducted with a Waters 410 GPC
The mixture of PEGDGE (0.005 mol), DEMC (0.005 mol) and
piperazine (0.01 mol) were reuxed in anhydrous ethanol (10
mL) for 24 h under nitrogen. Aer the reaction, the mixture was
poured into n-hexane and the precipitation was dried in vacuum
equipped with Waters Styragel column (HT4 + HT3) using
CDCl as the eluent, the molecular weights were calibrated with
polystyrene standards, and the ow rate was set at 1.0 mL min
at 35 C. FT-IR spectra were recorded by the US Nicolet com-
pany's AVATAR360 type collector, collection range: 4000–400
cm . DLS measurements were performed by Malvern Zetasizer
Nano ZS90, and the scattering angle was xed at 90 . TEM
3
ꢀ1
1
to get PEAC. H NMR (400 MHz, DMSO): d 6.24 (t, J ¼ 9.9 Hz,
ꢁ
ꢀ1
1
3
H), 6.16 (d, J ¼ 2.2 Hz, 1H), 5.84 (s, 1H); FTIR (neat, cm
)
400, 1732, 1616, 1110.
ꢀ
1
ꢁ
Preparation of PEAC/diPEAC micelles
studies were performed on a JEM-1011 electron microscope
operating at an acceleration voltage of 100 kV. Samples were
prepared by drop-casting onto Formvar Support Films and then
air-dried at room temperature before measurement. The
amount of the released DOX was measured using UV-vis spec-
troscopy (UV-2450PC, Shimadzu) at 480 nm wavelength and
calculated on the basis of following calibration curve 1 (pH 7.4),
calibration curve 2 (pH 5.0) and calibration curve 3 (in DI water)
PEAC is an amphiphilic polymer, and can be dispersed directly
in water to form micelles. 100 mg PEAC was added into 20 mL
DI water and stirred for 30 minutes. Then micellar aqueous
solution were obtained (5 mg mL ) with coumarin (hydro-
phobic segment) as the core and PEG (hydrophilic segment) as
the shell.
ꢀ1
Photo-dimerization crosslinked micelles (diPEAC) were
prepared by the following method. PEAC (50 mg) was dissolved
in DI water (50 mL) at room temperature. The solution was
subjected to UV-irradiation using a UV light (UV LED Glue
(1) Curing Machine, UPUL008, Japan) at an intensity of 75 mW
ꢀ1
using different concentrations of free DOX (2.5–100 mg mL ) in
the same buffer solution:
2
y ¼ 0.0289x + 0.0259, R ¼ 0.9995
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 105880–105888 | 105881

View Article Online
RSC Advances
Paper
ꢀ
2
cm at 365 nm. To study the cross-linking kinetics of the DLE (wt%) ¼
micelle, a certain amount of sample solution was made from
the irradiated solution at predetermined intervals and sub-
jected to UV-vis observation. UV-vis absorption spectra were
recorded on a Shimadzu UV-2401PC UV-vis spectrophotometer
with a 1.0 cm path length quartz cell.
The photo-dimerization degree (PD) was calculated from the
UV-vis spectra by comparing the peak absorption at 320 nm
assigned to the coumarin by the following equation:
(weight of loaded drug/weight of drug in feed) ꢂ 100% (5)
Photo-cross-linking of the DOX-loaded micelles (diPEAC-
DOX)
The cross-linked DOX-loaded micelles were prepared by the
same protocol. Freeze-dried PEAC micelle powder (50 mg) was
dissolved in 50 mL of water at room temperature. And then the
solution was subjected to UV-irradiation using a UV light. The
PD (%) ¼ [A ꢀ A ]/A
0
t
0
0 t
here, A and A
are the peak absorptions centered at 320 nm photo-cross-linking process was tracked by UV-vis.
assigned to the coumarin. 0 and t represent before irradiation
and aer the t time of irradiation by alternating light of 365 nm, In vitro DOX release
38
respectively.
The freeze-dried DOX-loaded PEAC was dissolved in phosphate
saline buffer (pH 7.4) and acetate buffer (pH 5.0) at a concentration
ꢀ1
Critical micelle concentration (CMC) measurements
of 1 mg mL . The above mixture was transferred into a dialysis
bag with a molecular weight cutoff of 3500 Da. The bag was then
immersed into a container with 20 mL of buffer solution at the
same pH value as that inside the bag. The outer phase of the buffer
Steady state uorescence spectra were obtained by a Perkin-
Elmer LS50B luminescence spectrometer. The PEAC solutions
ꢀ
1
with various concentrations from 0.0006 to 1 g L were added
to a series of volumetric asks. The emission wavelength was set
at 320 nm for uorescence excitation spectra. The spectra were
ꢁ
solution was oscillated at 37 C (50 rpm). At selected time intervals,
3
mL of the external buffer was withdrawn for UV-vis analysis and
ꢀ
1
replaced with the same amount of fresh buffer solution. The
released amount of DOX was determined from the absorbance at
recorded at a scan rate of 500 nm min . Excitation and emis-
sion slit widths are 0.75 nm, emission spectra recorded between
480 nm with the help of a calibration curve of DOX in the same
350 and 600 nm generated.
buffer. Then the accumulative weight and relative percentage of
the released DOX were calculated as a function of incubation time.
Evaluation of PEAC/diPEAC stability in the fetal bovine serum
The PEAC/diPEAC solutions were mixed with equal volumes of
PBS solution (pH 7.4, 0.01 M) containing 10% fetal bovine
serum (FBS, GIBCO) and incubated at 37 C. At various time
Cell lines
ꢁ
Two cell lines, HeLa (cervical cancer cells, human) and HepG2
(
Hepatocellular carcinoma cells, human), supplied by the
points, 5 mL aliquots of the solutions were removed and
analyzed by DLS.
Medical Department of Jilin University, China, were chosen for
cell tests. HeLa and HepG2 cells were cultured in Dulbecco's
modied Eagle's medium (DMEM, GIBCO) supplied with 10%
heat-inactivated FBS 2 mM L-glutamine, 100 U mL penicillin,
and 100 mg mL streptomycin (Sigma), at 37 C in a humidied
Preparation of DOX-loaded micelles (PEAC-DOX)
ꢀ
1
DOX-loaded PEAC was prepared by a straightforward method.
Briey, 20 mg DOX$HCl and 20 mL TEA were dissolved in DI
water (5 mL), stir for 30 minutes, then 200 mg PEAC was added
to the solution. Aer being stirred for an additional 2 h, 15 mL
water was added slowly to the mixture, dropwise, under
magnetic stirring at the same time. Aer stirred for another 2 h,
the mixture was transferred to a 3500 Da molecular weight
cutoff dialysis bag and dialyzed for 48 h to get rid of the free
DOX. The water was replaced every 8 h, and nally, the mixture
in the dialysis bag was freeze-dried to give red sponge-like
micelles. To determine the drug loading content, the DOX-
loaded PEAC was dissolved in DI water. The UV absorbance at
ꢀ1
ꢁ
2
atmosphere containing 5% CO .
Cytotoxicity
The cytotoxicity of micelles was examined by MTT assay. All sample
solutions were diluted with DMEM to obtain preset concentra-
tions. HeLa and HepG2 cells were seeded into 96-well plates with
a density of 104 cells per well and incubated in DMEM (100 mL) for
2
4 h. Then six concentrations (1.0, 0.5, 0.25, 0.12, 0.06 and 0.03 mg
ꢀ
1
mL ) of PEAC and diPEAC micelles were added to the wells, and
three parallel wells for each sample were used at a specic
concentration. Aer co-incubation with cells for 48 h, 20 mL of MTT
solution in PBS (5 mg mL ) was added to each well and the plate
was incubated for another 4 h at 37 C. Aer that, the medium
480 nm was measured to determine the DOX concentration
ꢀ1
based on the standard calibration curve obtained from free DOX
in DI water.
ꢁ
containing MTT was removed, and 150 mL of DMSO was added to
each well to dissolve the MTT formazan crystals. Finally, the plates
were shaken for 5 min, and the absorbance of formazan product
was measured at 490 nm by a Bio-Rad 680 microplate reader.
For anticancer activity analyses, we also use MTT assay to
4) evaluate the cytotoxicity of DOX-loaded PEAC/diPEAC micelles
Drug loading content (DLC) and drug loading efficiency
DLE) were calculated according to the following formula:
2
(
DLC (wt%) ¼
weight of loaded drug/weight of drug loaded micelles) ꢂ 100%
(
(
105882 | RSC Adv., 2015, 5, 105880–105888
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
and free DOX against HepG2 cells with different DOX dosages
PEAC was synthesized from DEMC, PEGDGE and Pip by
nucleophilic addition/ring-opening reactions as shown in
Scheme 1. The polymerization reaction is simple, mild and no
small molecules were generated during the polymerization with
ꢀ
1
from 0.001 to 100 mg mL
.
Cellular uptake studies
39
the feature of “click-chemistry”. GPC was utilized to determine
n w n
the molecular weights M and polydispersity (M /M ) which
Cellular uptakes by HepG2 cells were examined using confocal
laser scanning microscope (CLSM). HepG2 cells were seeded in
4
were 2.84 ꢂ 10 and 1.96, respectively. The structure of PEAC
1
was characterized by H NMR spectra (Fig. 1). The typical peaks
6
-well culture plates (a sterile coverslip was put in each well) at
5
of PEG and coumarin presented clearly in Fig. 1A. The chemical
shis at 6.0, 6.4 and 6.5 ppm in CDCl3 were pointed to the
proton of coumarin (H1–3).
a density of 1 ꢂ 10 cells per well and allowed to adhere for 24 h.
ꢀ
1
Then the cells were treated with free DOX (5 mg mL ) or DOX-
ꢀ1
loaded PEAC/diPEAC (5 mg mL , equivalent DOX concentra-
tion). Aer incubation for 0.5 h, 4 h and 24 h at 37 C, the
ꢁ
FTIR spectra of DHMC, DEMC and PEAC were shown in
Fig. 2. A strong stretching vibration of hydroxyl peaks at 3400
supernatant was carefully removed and the cells were washed
three times with ice-cold PBS and xed with 4% formaldehyde.
Aer the nucleus was stained with DAPI, the slides were
mounted. CLSM images were captured via confocal microscope
ꢀ
1
cm can be seen in PEAC and DHMC, but no corresponding
absorption peak in DEMC, because PEAC and DHMC contains
hydroxyl structure and none in DEMC. In addition, compared
ꢀ
1
with PEGDGE, new peaks at 1722 and 1616 cm assigned to
stretching vibration of C]O, and the aromatic ring of the
coumarin moieties appear in the FTIR spectrum of PEAC,
showing the successful introduction of coumarin into PEAC.
(Carl Zeiss LSM 710) under the same conditions.
Results and discussion
Synthesis of PEAC
Coumarin monomer with two epoxy groups (DEMC) was ob-
1
tained via two steps reaction. Its structure was conrmed by H
1
3
NMR, C NMR and FTIR as show in Fig. S1 and S2† and Fig. 2,
respectively. In Fig. S1 and S2A,† all proton atoms correspond-
ing to their characteristic chemical shis can be found. The
chemical shis at 6.0, 6.4 and 6.5 ppm (Ha–c) were the typical
peaks of coumarin. And in Fig. S2B,† the ve signals at 161.9,
1
60.0, 158.4, 156.6 and 154.5 ppm can be unambiguously
assigned to C1–5 and the four signals at 111.5, 104.7, 97.3 and
5.0 ppm were explicitly assigned to C6–9, nine signals were the
9
characteristic peak of the benzo pyran ring in coumarin. Two
signals at 49.8 and 44.3 ppm are the characteristic peaks of the
epoxide group.
Fig. 2 FTIR characterization of DHMC, DEMC and PEAC.
1
36,37
Fig. 1 H-NMR of PEAC in CDCl
3
(A) and in D
2
O (B).
Scheme 1 Synthetic route for PEAC.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 105880–105888 | 105883

View Article Online
RSC Advances
Paper
Coumarin contain a strongest ultraviolet absorption peak
around 320 nm, as shown in Fig. 3, both DEMC and PEAC has
a maximum absorption peak at about 320 nm demonstrated
that PEAC contains coumarin moieties.
PEAC could form the micelles in aqueous solution by
dispersing in water without any organic solvent. Then concen-
ꢀ
1
tration of PEAC could reach to 10 mg mL in water. In Fig. 1B,
these peaks for coumarin almost disappeared in D O, revealing
2
that PEAC formed micelles with almost all the coumarin moiety
transferring to the inner cores. Coumarin has a benzo pyran
ring, which is a very good uorescence emission group. So this
type of polymer does not require additional uorescence probe
such as pyrene to detect the critical micelle concentration. The
CMC of PEAC was calculated directly by using the uorescence
of coumarin according to the ref. 38 and 40. As shown in Fig. 4,
ꢀ3
ꢀ1
the CMC value was calculated to be 4.0 ꢂ 10 g L for PEAC.
Coumarin is a light-sensitive materials, photo-dimerization
occur under irradiation at 365 nm. As illustrated in Scheme 2.
PEAC was dispersed in DI water with the concentration of
ꢀ
1
1
g L . Under illumination at 365 nm for different time,
Scheme
2
Schematic illustration of micellization of PEAC, the
formation of cross-linked micelles under UV-irradiation and DOX-
loaded micelles.
nanoparticles with different degree of cross-linking could be
obtained. The process of cross-linking is monitored by the UV-
vis spectrophotometer. As shown in Fig. 5, with the increase of
exposure time, maximum absorption intensity at 320 nm
decreased gradually, indicating the occurrence of the light
dimerization. The photo-dimerization degree of PEAC increased
to 50% rapidly in the rst 5 minutes and increased to 70% in the
next 10 minutes.
DLS was utilized to study the size distribution of the
micelles. The average diameter of PEAC micelle was 110 nm
(
(
(
Fig. 6A). Aer photo-cross-linking, the size changed to 85 nm
Fig. 6B). The diameter of PEAC-DOX micelle was 140 nm
Fig. 6C) and changed to 120 nm (Fig. 6D) aer photo-cross-
Fig. 3 UV-vis characterization of DEMC and PEAC.
linking. The shrinkage of the micelles was ascribed to the
cross-linking. This shrinkage was conrmed by TEM, the mean
diameter of spherical micelles before and aer photo-cross-
linking was about 50 nm (Fig. 6E) and 40 nm (Fig. 6F), respec-
tively. For DOX-loaded PEAC micelles, the diameter is about 70
nm (Fig. 6G) and it changed to 60 nm (Fig. 6H) aer cross-
linking. Moreover, all particles presented good dispersibility,
even the cross-linked particles, implying that there was no
micelle aggregation during the process of cross-linking. Particle
size measured by TEM is smaller than the DLS, mainly due to
the volume shrinkage in the drying process in the preparation
41,42
of the TEM sample.
PEAC micelles, the diameter are about 70 nm (Fig. 6G) and
changed to 60 nm (Fig. 6H) aer cross-linking. Moreover, all
particles presented good dispersibility, even the cross-linked
particles, implying that there was no micelle aggregation
during the process of cross-linking. Particle size measured by
TEM is smaller than the DLS, mainly due to the volume
Fig. 4 Intensity from the fluorescence excitation spectra with PEAC in
different concentration. Inset: fluorescence emission spectra with
different PEAC concentration.
105884 | RSC Adv., 2015, 5, 105880–105888
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Fig. 5 UV-vis spectra of polymer nanoaggregates upon 365 nm irra-
diation with different time (A), photo-dimerization degree of PEAC as
a function of irradiation time based on the absorbance change at 320
nm (B).
Fig. 6 DLS results of PEAC micelles (A) and diPEAC (B); PEAC-DOX (C)
and diPEAC-DOX (D); TEM images of PEAC micelles (E) and diPEAC (F);
PEAC-DOX (G) and diPEAC-DOX (H) (bar ¼ 500 nm).
shrinkage in the drying process in the preparation of the TEM
41,42
sample.
Stability of PEAC/diPEAC micelles
The stability of the PEAC/diPEAC micelles was studied in PBS
ꢁ
(
pH 7.4) containing 10% FBS at 37 C by monitoring the particle
size change as a function of time. As it was shown in Fig. 7, there
was no signicant change in size during 24 h, suggesting that
neither aggregation nor destabilization was induced by serum
proteins.
Drug loading and release in vitro
To assess the inuence of tertiary amino groups and photo-
dimerization degree (%) on drug incorporation, DOX was
used as a model anticancer drug to evaluate the drug loading
and release properties. Drug-loading was done by directly
dispersing drug and PEAC together into water without any
organic solvents or auxiliaries. The drug loading contents (DLC)
and the drug loading efficiency (DLE) of PEAC-DOX were
measured to be about 6.78 wt% and 67.8% (the theoretical DLC
Fig. 7 Particle size and PdI of micelles in PBS (pH 7.4) containing 10%
FBS at 37 C for different time periods determined by DLS.
ꢁ
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 105880–105888 | 105885

View Article Online
RSC Advances
Paper
was designed to be 10 wt%). It is worthy to mention that photo-
dimerization of micelles under UV irradiation at 365 nm did not
affect both DLC and DLE.
The in vitro DOX release from PEAC and diPEAC was con-
ducted in two different buffered solutions (pH 7.4 and 5.0) at
ꢁ
3
7 C. As shown in Fig. 8, dramatic burst release of DOX was not
observed for both PEAC and diPEAC micelles at pH 7.4 and pH
.0, demonstrating DOX was well entrapped in the inner core of
5
both micelles. Meanwhile, the release of DOX from diPEAC
micelles was signicantly slower than that from PEAC micelles.
At pH 7.4 (Fig. 8A), only approximately 11% of DOX was released
during 60 h from diPEAC micelles (photo-dimerization degree
was 80%), while only 20% of DOX was released from the PEAC
micelles under the same conditions. The same phenomenon
can be seen at pH 5.0 (Fig. 8B). The release amount was about
20% from diPEAC micelles for 60 hours, compared with a 50%
release of PEAC-DOX micelles. These data also indicated that
the both micelles have pH-sensitivity (lower drug release at pH
7.4 and higher drug release at pH 5.0). This character would be
useful for drug delivery to obtain a long-term circulation in
blood and an effective drug release at the tumor site, since the
microenvironment around the tumor cells and inside the
endosome is acidic.
At the same time, we noticed that the degree of cross-linking
showed a certain inuence on the release rate. At pH 7.4, when
the photo-dimerization degree is 50%, the release drug was
about 15%. It was obvious the release rate decreases with the
increase of photo-dimerization degree, because the cross-
linking made the micellar core more compact, and sup-
pressed the release of DOX from the micelles. The sustained
release of DOX from the diPEAC micelles is conducive to the
extension of drug circulation time and protects the drug from
enzyme degradation, rapid renal clearance and interactions
with serum proteins and thus would signicantly enhance drug
2
delivery to tumors.
Cell internalization
The cellular uptake and intracellular release behaviors of free
DOX and DOX-loaded PEAC/diPEAC were investigated by CLSM.
Free DOX and DOX-loaded PEAC/diPEAC were incubated with
ꢁ
HepG2 cells at 37 C for 0.5 h, 4 h and 24 h. As shown in Fig. 9,
intracellular distribution of DOX-loaded PEAC/diPEAC was
different from that of free DOX. Red uorescence was observed
mainly in the cellular nuclei aer 0.5 h of incubation with free
Fig. 9 CLSM-images of HepG2 cells incubated with free DOX and
DOX-loaded PEAC/diPEAC micelles for 0.5 h, 4 h, and 24 h. For each
row, images from left to right were the cells with nucleus stained with
DAPI (blue), with DOX (red) fluorescence, and overlapped images (bar
¼
20 mm).
DOX (Fig. 9A). However, when the cells were incubated with
DOX-loaded micelles, the uorescence was observed mainly in
Fig. 8 In vitro release profiles of DOX from PEAC and diPEAC micelles the cytoplasm rather than the cell nuclei (Fig. 9D and G). Aer 4
ꢁ
at pH 7.4 (A) and pH 5.0 (B) at 37 C (50%, 60%, 70%, 80% point to
h incubation, the uorescence in the nuclei of the cells became
different photo-dimerization degree (%)).
105886 | RSC Adv., 2015, 5, 105880–105888
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
stronger for free DOX (Fig. 9B), while the cells incubated with
DOX-loaded micelles for 4 h emitted increased uorescence in
the cytoplasm as well as the very weak uorescence in nuclei
(Fig. 9E and H). When the incubation period increased to 24 h,
the uorescence in the nuclei of the cells became weaker for free
DOX (Fig. 9C). For DOX-loaded micelles, the red uorescence
nuclei increased with time (Fig. 9F and I). These data suggest
that the internalization mechanism of DOX-loaded micelles is
different from that of free DOX. On the other hand, the uo-
rescence intensity of the diPEAC micelles (Fig. 9G–I) was much
stronger than the PEAC micelles (Fig. 9D–F).
The smaller particle size and the more stable structure of the
photo-cross-linked micelles should take some responsibility for
43,44
Fig. 11 Cell viability of HepG2 cells against DOX, DOX-loaded PEAC/
diPEAC micelles after cultured for 48 h with different DOX dosages.
this result.
DOX-loaded micelles will eventually enter the
nuclei where DOX interacts with topoisomerase II to cause DNA
2
cleavage and cytotoxicity, as the following cytotoxicity results
demonstrate that the growth of HepG2 cells can be effectively
inhibited (Fig. 8).
diPEAC micelles, which had been seened in the previous liter-
45,46
ature.
The lower cytotoxicity of DOX-loaded micelles can be
In order to check whether the micelles go (even partly) to the
lysosomes or not, HepG2 cells incubated with Calcein loaded-
attributed to the slow release of DOX from micelles as evidenced
by the in vitro release in Fig. 8 and delayed nuclear uptake in
HepG2 cells which has been proved by internalization studies
by CLSM (Fig. 9).
ꢁ
micelles for 1 h at 37 C at a concentration of 10 mM. The cal-
cein uorescence was encapsulated into micelles instead of
DOX. As shown in Fig. S3,† lysosomes stained with Lyso-Tracker
Red (red uorescence) was mostly overlapped with Calcein

uorescence (green), indicating that the micelles go mostly to
Conclusions
the lysosomes.
In summary, a coumarin-based pH-responsive copolymer was
successfully synthesized. The core of the polymeric micelles was
cross-linked by UV-irradiation at 365 nm via photo-dimerization
of the coumarin moiety to form the cross-linked micelles. Drugs
can be loading easily during the self-assembly of copolymers.
The PEAC and diPEAC micelles both have pH-sensitivity, low
drug release at pH 7.4 and high drug release at pH 5.0. In
addition, the cross-linked micelles had slower drug release in
comparison with the uncross-linked micelles. The cross-linked
DOX-loaded micelles could be successfully internalized by
cancer cells, showing high cellular uptake efficiency. All in all,
we are convinced that the cross-linked DOX-loaded micelles
would have a great potential in cancer therapy applications.
Cytotoxicity
MTT assay was applied to evaluate the cytotoxicity of the blank
polymer micelles by using HeLa and HepG2 cells. Aer incu-
bation with PEAC and diPEAC micelles at gradient concentra-
tions from 1.0 to 0.03 mg mL for 24 h, cells showed high
viability (80% and above) even the concentrations of micelle up
to 1 mg mL , displaying that both micelles have good
biocompatibility and safe to be used as drug carriers (Fig. 10).
The in vitro cytotoxicities of DOX-loaded micelles and the
free DOX were evaluated by MTT assay against HepG2 cells with
different DOX dosages from 0.001 to 100 mg mL . As shown in
Fig. 11, the half maximal inhibitory concentration (IC50) values
for free DOX, PEAC-DOX and diPEAC-DOX against HepG2 cell
lines were 0.56, 2.84 and 4.43 mg mL , respectively. These
results demonstrated that DOX-loaded micelles were able to
enter the cells and exhibited a suitable pharmacological effect
on cancer cells. Meanwhile, free DOX showed higher inhibition
for cancer cell proliferation than that of the DOX-loaded PEAC/
ꢀ
1
ꢀ
1
ꢀ1
ꢀ1
Acknowledgements
The authors would like to thank the nancial support from
National Natural Science Foundation of China (No. 51203043,
No. 51403051, No. 21502042) and Developing Program of
Natural Science of Henan province (No. 142300410122).
Notes and references
1
2
3
4
L. S. Yan, L. X. Yang, H. Y. He, X. L. Hu, Z. G. Xie, Y. B. Huang
and X. B. Jing, Polym. Chem., 2013, 3, 1301.
H. H. Kuang, H. Y. He, J. Hou, Z. G. Xie, X. B. Jing and
Y. B. Huang, Macromol. Biosci., 2013, 13, 1593.
C. C. Lee, J. A. Mackay, J. M. Fr ´e chet and F. C. Szoka, Nat.
Biotechnol., 2006, 23, 1517.
Fig. 10 Cell viability of HeLa and HepG2 cells incubated with PEAC
and diPEAC micelles. Data were presented as mean ꢃ standard devi-
ation (n ¼ 3).
W. B. Liechty and N. A. Peppas, Eur. J. Pharm. Biopharm.,
2012, 80, 241.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 105880–105888 | 105887

View Article Online
RSC Advances
Paper
5
H. S. Han, K. Y. Choi, H. Ko, J. Jeon, G. Saravanakumar, 25 A. Elena, M. M. Dolores, M. M. E. Ram ´o n, S. F ´e lix, S. Juan,
Y. D. Suh, D. S. Lee and J. H. Park, J. Controlled Release,
014, 200, 158.
P. H. Edgar and F. M. Alberto, Eur. J. Pharm. Biopharm., 2015,
3, 52.
A. Pedro and G. Carmen, J. Am. Chem. Soc., 2009, 131, 6833.
26 J. Li, S. Guo, M. Wang, L. Ye and F. Yao, RSC Adv., 2015, 5,
19484.
2
6
7
9
27 M. Nagata and Y. Yu, React. Funct. Polym., 2008, 68, 915.
G. Gaucher, M. H. Dufresne, V. P. Sant, N. Kang, 28 S. R. Trenor, A. R. Shultz, B. J. Love and T. E. Long, Chem.
D. Maysinger and J. C. Leroux, J. Controlled Release, 2006,
09, 169.
Z. S. Ge and S. Y. Liu, Chem. Soc. Rev., 2013, 42, 7289.
Rev., 2004, 104, 3059.
29 H. B. Sun, H. L. Fan and X. H. Peng, J. Org. Chem., 2014, 79,
1
8
9
1
11359.
C. Wang, L. Cheng and Z. Liu, Biomaterials, 2011, 32, 1110. 30 F. Borges, F. Roleira, N. Milhazes, L. Santana and E. Uriarte,
Curr. Med. Chem., 2005, 12, 887.
P. T. Hammond and Y. Y. Yi, Adv. Drug Delivery Rev., 2011, 31 G. Xu, C. Shi, D. Guo, L. Wang, Y. Ling, X. Han and J. Luo,
3, 1228. Acta Biomater., 2015, 21, 85.
1 H. Cabral and K. Kataoka, J. Controlled Release, 2014, 190, 32 J. Qiao, M. J. Samarendra and A. Seema, Polym. Chem., 2012,
65. 3, 2785.
2 W. T. Lu, X. X. Wang, R. Cheng, C. Deng, F. H. Meng and 33 C. Chen, X. Liu, J. Ji, G. Liu, S. Pang, C. Zhu and L. Lv, Polym.
Z. Y. Zhong, Polym. Chem., 2015, 6, 6001. Chem., 2011, 2, 1389.
3 Y. Q. Shen, J. Erlei, Z. Bo, M. Caitlin, J. M. H. Sui, J. Zhao, 34 M. J. Robb, L. A. Connal, B. F. Lee, N. A. Lynd and
0 V. Shrinivas, J. L. Hedrick, Y. O. Zhan, C. Yang, P. L. R. Ee,
6
1
1
1
4
J. Q. Wang, J. B. M. H. Fan and V. K. Edward, J. Am. Chem.
Soc., 2010, 132, 4259.
4 Y. Shen, Y. Zhan, J. Tang, P. Xu, P. A. Johnson, M. Radosz,
C. J. Hawker, Polym. Chem., 2012, 3, 1618.
35 N. M. Khan, H. Kapahi, S. Kumar, T. Bhardwaj, S. Arora and
N. Mishra, J. Drug Targeting, 2015, 23, 387.
1
1
1
E. A. Van Kirk and W. J. Murdoch, AIChE J., 2008, 54, 2979. 36 X. S. Jiang, R. Wang, Y. R. Ren and J. Yin, Langmuir, 2009, 25,
5 R. F. Pagels and R. K. Prud'homme, J. Controlled Release,
015, 219, 519.
6 A. R. Bilia, M. C. Bergonzi, C. Guccione, M. Manconi,
9629.
2
37 G. X. Fu, Z. L. Su, X. S. Jiang and J. Yin, Polym. Chem., 2014, 5,
2027.
A. M. Fadda and C. Sinico, J. Drug Delivery Sci. Technol., 38 J. Q. Jiang, Q. Z. Shu, X. Chen, Y. Q. Yang, C. L. Yi, X. Q. Song,
015, DOI: 10.1016. X. Y. Liu and M. Q. Chen, Langmuir, 2010, 26, 14247.
7 M. L. Wang, J. Sun, Y. L. Zhai, H. Lian, C. Luo, L. Li, Y. Q. Du, 39 H. Nandivada, X. Jiang and J. Lahann, Adv. Mater., 2007, 19,
2
1
1
1
D. Zhang, W. Ding and S. H. Qiu, Biomacromolecules, 2015,
6, 1179.
8 W. W. Du, Y. C. Fan, B. He, N. Zheng, L. Yuan, W. B. Dai,
H. Zhang, X. Q. Wang, J. C. Wang and X. Zhang, Mol.
Pharm., 2015, 12, 1467.
2197.
1
40 T. J. V. Prazeres, M. Beija, F. V. Fernandes, P. G. A. Marcelino,
J. P. S. Farinha and J. M. G. Martinho, Inorg. Chim. Acta,
2012, 381, 181.
41 K. Hayakawa, T. Yoshimura and K. Esumi, Langmuir, 2003,
19, 5517.
9 R. Ja-Hyoung, R. T. Chacko, J. Siriporn, B. Sean,
B. R. Prakash and S. Thayumanavan, J. Am. Chem. Soc., 42 C. Feng, Z. Shen, Y. G. Li, L. N. Gu, Y. Q. Zhang, G. L. Lu and
2
010, 132, 17227.
0 K. Alexander and V. Serguei, Angew. Chem., Int. Ed., 2009, 48,
418.
1 F. Ercole, T. P. Davis and R. A. Evans, Polym. Chem., 2010, 1,
7.
X. Y. Huang, J. Polym. Sci., Part A: Polym. Chem., 2009, 47,
1811.
43 S. H. Wu, H. H. Kuang, F. B. Meng, Y. G. Wu, X. Y. Li,
X. B. Jing and Y. B. Huang, J. Mater. Chem., 2012, 22, 15348.
44 Y. Kim, M. H. Pourgholami, D. L. Morris and M. H. Stenzel,
Biomacromolecules, 2012, 13, 814.
2
2
2
2
2
5
3
2 F. Jia, Y. Wang, H. B. Wang, Q. Jin, T. G. Cai, Y. J. Chen and
J. Ji, Polym. Chem., 2015, 6, 2069.
3 R. K. O'Reilly, C. J. Hawker and K. L. Wooley, Chem. Soc. Rev.,
45 X. T. Shuai, A. Hua, N. Nasongkla, S. Kim and J. M. Gao, J.
Controlled Release, 2004, 98, 415.
2
007, 38, 1068.
46 B. Younsoo, F. Shigeto, H. Atsushi and K. Kazunori, Angew.
Chem., 2003, 42, 4640.
4 R. Yang, F. H. Meng, S. B. Ma, F. S. Huang, H. Y. Liu and
Z. Y. Zhong, Biomacromolecules, 2011, 12, 3047.
105888 | RSC Adv., 2015, 5, 105880–105888
This journal is © The Royal Society of Chemistry 2015