Photo-responsive reversible micelles based on azobenzene-modified poly(carbonate)s via azide-alkyne click chemistry

Article abstract of DOI:10.1039/c4ra07345b

Photo-induced reversible amphipathic copolymer PMPC-azo was click conjugated by connecting amphiphilic poly(ethylene glycol)-modified poly(carbonate)s (PEG-b-poly(MPC)) and azide-functional trifluoromethoxy-azobenzene (azo-N₃). The resulting copolymer self-assembled into spherical micelles with a hydrophobic azo core stabilized by a hydrophilic PEG corona in aqueous solution. As characterized by time-resolved UV-vis spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM), these micelles showed reversible self-assembly and disassembly in aqueous solution under alternating UV and visible light irradiation. The model drug Nile Red (NR) was then successfully encapsulated into the micelles. Light-controlled release and re-encapsulation behaviors were demonstrated by fluorescence spectroscopy. The cell cytotoxicity of PMPC-azo micelles was also evidenced by MTT assay. This study provides a convenient method to construct smart nanocarriers for controlled release and re-encapsulation of hydrophobic drugs. This journal is

Full text of DOI:10.1039/c4ra07345b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Photo-responsive reversible micelles based on
azobenzene-modified poly(carbonate)s via azide–
alkyne click chemistry†
Cite this: RSC Adv., 2014, 4, 47929
*
Ding Hu, Yefei Li, Yile Niu, Ling Li, Jingwen He, Xiangyu Liu, Xinnian Xia, Yanbing Lu,
Yuanqin Xiong and Weijian Xu
Photo-induced reversible amphipathic copolymer PMPC-azo was click conjugated by connecting
amphiphilic poly(ethylene glycol)-modified poly(carbonate)s (PEG-b-poly(MPC)) and azide-functional
trifluoromethoxy-azobenzene (azo-N3). The resulting copolymer self-assembled into spherical micelles
with
a hydrophobic azo core stabilized by a hydrophilic PEG corona in aqueous solution. As
characterized by time-resolved UV-vis spectroscopy, dynamic light scattering (DLS) and transmission
electron microscopy (TEM), these micelles showed reversible self-assembly and disassembly in aqueous
solution under alternating UV and visible light irradiation. The model drug Nile Red (NR) was then
successfully encapsulated into the micelles. Light-controlled release and re-encapsulation behaviors
were demonstrated by fluorescence spectroscopy. The cell cytotoxicity of PMPC-azo micelles was also
evidenced by MTT assay. This study provides a convenient method to construct smart nanocarriers for
controlled release and re-encapsulation of hydrophobic drugs.
Received 20th July 2014
Accepted 5th September 2014
DOI: 10.1039/c4ra07345b
www.rsc.org/advances
disassembly behaviors via irradiating with UV and visible
light.24 Recently, Blasco et al. reported a series of light-respon-
Introduction
sive vesicles based on linear-dendritic amphiphiles, which
tailored the photo-responsive properties of the vesicles and
consequently the release rate via adjusting the percentages of
azo and hydrocarbon chains.25
During the last decades, stimuli-responsive block copolymers
(BCP) have been widely investigated for “on–off” drug delivery
and “on-demand” nanomedicines because of their switch
properties.1–5 The most frequently used stimuli are pH,
temperature, light, redox potential, ultrasound, charge, gases,
biomolecules and enzymes.6–14 Compared with other stimuli-
responsive systems, light-responsive BCP do not require any
changes in the surroundings. In addition, the wavelength and
intensity of illumination can be accurately adjusted and the
time, direction and area of illumination are easily
controlled.15–18 Light-responsiveness is usually provided by
photochromic molecules attached to the polymers. Azobenzene
(azo) is a well-known and well-used compound because it can
undergo trans–cis photo isomerization in response to UV and
visible light. Azo isomerization is a facile method for changing
the hydrophobicity of BCP micelles since the cis form of azo-
benzene is more polar than the trans form.15 There are several
reports on amphipathic BCP decorated with azobenzenes.19–23
Chen et al. reported the construction of photo-responsive
micelles from azobenzene-modied hyperbranched poly-
phosphates, which showed reversible self-assembly and
Amphiphilic copolymers have been widely studied for their
potential applications in biomedical and pharmaceutical elds.
Among them, block copolymers of aliphatic poly(carbonate)s
combined with poly(ethylene glycol) (PEG) have been widely
investigated for drug delivery because of their low toxicity,
biocompatibility, and biodegradability.26–30 PEG is a hydrophilic
and nonionic polymer that has been widely used as a biocom-
patible polymer in both academia and industry for chemical
and biological applications.31–33 Polymeric micelles containing
PEG as the hydrophilic shell are able to form a palisade pre-
venting the adsorption of proteins and enzymes and subse-
quent non-specic uptake by the reticuloendothelial system
(RES) aer intravenous injection.34,35 Several groups have graf-
ted PEG onto polyesters using different synthetic pathways.
Yang et al. reported the ring opening copolymerization of
acryloyl carbonate (AC) and 3-caprolactone (CL) using methoxy
PEG as an initiator.36 Hydrophilic and amphiphilic
PEG-poly(trimethylene carbonate) hydrogels were prepared
using PEG-bisazides via “click” chemistry by Truong et al.37
However, there have been limited reports for the synthesis of
stimuli-responsive poly(carbonate)s. Zhong's group prepared
pH-responsive biodegradable polymers, which were comprised
of a novel acid-labile polycarbonate hydrophobe and PEG.38
Institute of Polymer Science and Engineering, College of Chemistry & Chemical
Engineering, Hunan University, Changsha 410082, China. E-mail: yanbinglu@hnu.
edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra07345b
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 47929–47936 | 47929

View Article Online
RSC Advances
Paper
They also developed reduction-responsive poly-(carbonate)s via before measurement. The mean size of the micelles was deter-
ring-opening copolymerization of 3-CL and pyridyl disulde- mined by dynamic light scattering (DLS) using a Malvern Nano
functionalized cyclic carbonate monomers.39,40 However, there S instrument (Malvern, UK). Measurements of the solution of
is no report on light-responsive biodegradable biomaterials micelles (0.5 mg mLꢁ1) were performed at a scattering angle of
based on poly(carbonate)s.
90ꢂ and at room temperature (25 ꢂC). UV-vis spectra were
In this study, we developed a facile method to prepare carried out using a Hitachi UV-4100 UV-vis spectrophotometer
amphiphilic block copolymers with poly(carbonate)s as a and the spectra were collected within the range of 200–800 nm.
hydrophobic chain using a ring opening polymerization of
cyclic carbonates. Then, copper-catalyzed Huisgen 1,3-dipolar
cycloaddition of azides with alkynes was employed as the
Synthesis of 4-hydroxy-40-triuoromethoxyazobenzene (1)
coupling reaction to form reversible light-responsive biode- 4-(Triuoromethoxy)aniline (4.5 g, 25.4 mmol) was dissolved in
gradable micelles based on hydrophobic poly(carbonate)s. This 1.5 M aqueous H2SO4 (70 mL) and placed in an ice bath at 0 ꢂC.
amphiphilic copolymer can self-assemble into micelles con- Sodium nitrite (2.35 g, 34.0 mmol) in water (15 mL) was added
sisting of a hydrophobic core surrounded by a hydrophilic shell. dropwise to the former solution and stirred for 3 h. Then, the
Such core–shell polymeric micelles allow the properties of mixture was added dropwise to a solution of sodium carbonate
reversible transition via irradiation with UV and visible light. (15.7 g, 147.9 mmol), sodium hydroxide (1.0 g, 25.0 mmol) and
The micelle formation, reversible light-responsive self-assembly phenol (2.5 g, 26.6 mmol) in water (100 mL) at 0 ꢂC and stirred
and disassembly of the micelles, and cytotoxicity of the copol- for 3 h. The resultant mixture was poured into deionized water,
ymer micelles, as well as light-responsive model drug release and then neutralized with 2 M aqueous HCl. The crude product
were investigated.
was ltered and washed with a large volume of deionized water,
and then dried in a vacuum oven at 60 ꢂC. Yield: 5.7 g (79.6%).
1H NMR (CDCl3): d ¼ 6.95–7.94 (m, 8H, ArH), 5.30 (s, 1H,
ArOH);
Experimental section
Materials
13C NMR (CDCl3): d ¼ 158.50, 150.93, 150.45, 146.99, 125.15,
123.99, 121.39, 115.87.
Monomethoxy poly(ethylene glycol) (PEG5k, Mn ¼ 5000), 4-(tri-
uoromethoxy)-aniline, ethyl chloroformate, 1,4-dibromobu-
tane and 2,2-bis(hydroxyl methyl) propionic acid were obtained
from Sigma-Aldrich and used as received. Dichloromethane
(DCM), dimethylformamide (DMF), 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU), and triethylamine were dried over calcium
hydride for 24 h at room temperature and distilled under
reduced pressure. The thiourea catalyst (TU) was synthesized as
previously reported and recrystallized from dry DCM.26,41
Tetrahydrofuran (THF) was dried by reuxing over a benzo-
phenone–sodium mixture until a deep blue color appeared and
distilled. All the other reagents and solvents were purchased
from Sinopharm Chemical Reagent Co. Ltd, China and used as
received.
Synthesis of 4-(4-bromobutyloxy)-40-triuoromethoxy-
azobenzene (2)
To a 100 mL three-neck round bottomed ask was added 1,4-
dibromobutane (12.9 g, 60 mmol), K2CO3 (2.76 g, 20 mmol), KI
(0.12 g, 0.7 mmol) and acetone (30 mL). To a 50 mL pressure
equalizing dropping funnel, compound 1 (5.6 g, 20 mmol) in 40
mL acetone was added. The solution was added dropwise to the
ask. The reaction mixture was magnetically stirred at 60 ꢂC for
12 h. The salts formed were ltered and washed with acetone.
The solvent of the ltrate was evaporated using a rotary evap-
orator. The crude product was washed with a very small amount
of ethanol and chloroform, and then recrystallized from
methanol and dried in a vacuum oven at 50 ꢂC. Yield: 5.8 g
(69.5%).
Characterization
1H and 13C NMR spectra were recorded on an INOVA-400 NMR
spectrometer in chloroform-d (CDCl3). Mass spectroscopy 2H, CH2CH2CH2CH2Br), 3.50–3.53 (t, 2H, CH2CH2CH2CH2–Br),
analyses were conducted using an LCQ-Advantage mass spec- 1.96–2.14 (m, 4H, CH2CH2CH2CH2Br);
trometer (Thermo Finnigan, United States). The number-
1H NMR (CDCl3): d ¼ 6.99–7.94 (m, 8H, ArH), 4.08–4.11 (t,
13C NMR (CDCl3): d ¼ 161.74, 151.11, 150.38, 146.99, 125.07,
average molecular weight (Mn) and molecular distribution 124.08, 121.27, 114.72, 67.29, 33.41, 29.62, 27.81.
(polydispersity index, PDI ¼ Mw/Mn) of the polymers were
determined at room temperature using a Waters GPC (Waters,
Synthesis of 4-(4-azidebutyloxy)-40-triuoromethoxy-
azobenzene (azo-N3)
USA) equipped with four TSK HXL series polystyrene divinyl-
benzene gel columns (300 ꢀ 7.8 mm). Calibration was estab-
lished with polystyrene standards from Polymer Laboratories. Compound 2 (5 g, 12.0 mmol) and sodium azide (1.56 g, 24.0
THF was used as solvent with a ow rate of 1 mL minꢁ1
.
mmol) were dissolved in DMF (15 mL), and the mixture was
Transmission electron microscopy (TEM) images were obtained stirred at room temperature for 24 h. The solution was added to
on an H-7000 NAR transmission electron microscope (Hitachi) DCM (100 mL) and reverse-extracted three times with water (100
with a working voltage of 100 kV. A drop of the micelle aqueous mL ꢀ 3). The organic phase was dried with anhydrous magne-
solution (0.5 mg mLꢁ1) was deposited onto a 230 mesh copper sium sulfate, ltered and evaporated to dryness. The product
grid coated with carbon and allowed to dry at room temperature was collected as a yellow powder. Yield: 4.1 g (90.1%).
47930 | RSC Adv., 2014, 4, 47929–47936
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
1H NMR (400 MHz, CDCl3): d ¼ 7.01–7.96 (m, 8H, ArH), 4.09–
1H NMR (400 MHz, CDCl3): d ¼ 4.71 (m, OCH2CCH), 4.25–
4.12 (t, 2H, CH2CH2CH2CH2N3), 3.40–3.43 (t, 2H, CH2CH2- 4.33 (m, OC(O)OCH2), 3.62 (m, OCH2CH2O), 3.36 (s, CH3O), 2.53
CH2CH2N3), 1.81–1.98 (m, 4H, CH2CH2CH2CH2N3);
13C NMR (400 MHz, CDCl3): d ¼ 161.66, 151.10, 150.39,
147.00, 125.36, 124.06, 121.27, 114.72, 67.63, 51.01, 26.44,
25.57.
(s, CH2CCH), 2.30 (br s, OH), 1.27 (s, CH3).
GPC (THF, RI): Mn (PDI) ¼ 10 544 g molꢁ1 (1.11).
Synthesis of PMPC-azo via “click” chemistry
In a Schlenk tube, PEG-b-poly(MPC) (302.9 mg, propargyl group,
0.74 mmol), azo-N3 (289.2 mg, 0.74 mmol), sodium ascorbate
(17.1 mg, 0.074 mmol), and DMF (4 mL) were introduced. The
tube was tted with a rubber septum. The solution was further
degassed using three freeze–pump–thaw cycles. A DMF solution
of copper sulfate (10.55 mg, 0.037 mmol) was then added to the
Schlenk tube. The solution was stirred at room temperature for
24 h. The crude material was puried by dialysis (dialysis tubing
3500 MWCO) against deionised water, which was regularly
renewed. Aer 3 days, the nal product, PMPC-azo, was
obtained by lyophilization. Yield: 450.0 mg (76.0%).
Synthesis of 2,2-bis(hydroxyl methyl)propionate (3)
In a 250 mL round-bottom ask, 2,2-bis(hydroxyl methyl)-pro-
pionic acid (19.21 g, 143.3 mmol), KOH (8.6 g, 153.6 mmol) and
DMF (100 mL) were added. The mixture was stirred at 100 ꢂC for
2 h, and then propargyl bromide (18.28 g, 153.6 mmol) was
added dropwise over 30 min. Aer 72 h of reaction, the reaction
mixture was ltered, the solvent was evaporated under reduced
pressure, and the residues were dissolved in 200 mL of DCM
and extracted three times with saturated salt water (100 mL ꢀ 3).
The organic phase was concentrated to yield the crude product,
which was puried by column chromatography (eluent: ethyl
acetate–petroleum ether ¼ 1/5, v/v). Yield: 10.68 g (43.4%).
1H NMR (400 MHz, CDCl3): d ¼ 4.76–4.77 (d, 2H, CHCCH2-
CO), 3.91–3.94 (d, 2H, CH2OH), 3.72–3.75 (d, 2H, CH2OH), 2.51–
2.52 (t, 1H, CHCCH2CO), 1.11 (s, 3H, CH3CC).
1H NMR (400 MHz, CDCl3): d ¼ 7.84–7.87 (m, ArH), 7.69 (br s,
N3CHC), 7.28–7.30 (d, ArH), 6.92–6.94 (d, ArH), 5.24 (s, C(O)
OCH2), 4.43 (m, NCH2), 4.24 (m, OC(O)OCH2), 4.02 (m, CH2-
CH2OAr), 3.64 (m, OCH2CH2O), 3.38 (s, CH3O), 2.11 (m,
NCH2CH2CH2CH2), 1.82 (m, NCH2CH2CH2CH2), 1.20 (s, CH3).
GPC (THF, RI): Mn (PDI) ¼ 16 285 g molꢁ1 (1.12).
13C NMR (400 MHz, CDCl3): d ¼ 175.01, 75.20, 67.82, 52.43,
49.29, 16.95.
Preparation of micelles
Synthesis of 5-methyl-5-propargylxycarbonyl-1,3-dioxane-2-
one (MPC)
Micelles of PEG-b-poly(MPC) and PMPC-azo were prepared by a
dialysis method. 10.0 mg copolymer was dissolved in DMF
(2 mL), and then DI water (20 mL) was slowly added with
vigorous stirring. Aer vigorous stirring for another 2 h at room
temperature, the micelles were obtained and further dialyzed
against DI water for 24 h to remove DMF (MWCO 1000 Da). The
nal polymer concentration was adjusted by adding DI water to
Compound 3 (8.60 g, 50 mmol) was mixed with ethyl chlor-
oformate (10.86 g, 100 mmol) and THF (100 mL) in a sealed
vessel that was purged with nitrogen and cooled in an ice bath.
Aer stirring for an hour, triethylamine (10.14 g, 100 mmol) was
added dropwise over 30 min under a nitrogen atmosphere. The
reaction was allowed to stir for 3 h, aer which it was allowed to
warm to 25 ꢂC and stirred overnight. The solution was then
ltered and evaporated to dryness, and the product was
precipitated in a mixture of ethyl acetate and diethyl ether (1 : 1)
as white crystals. Yield: 9.24 g (93.3%).
0.5 mg mLꢁ1
.
Fluorescence measurement of the critical micellar
concentration
The critical micellar concentrations (CMC) of PEG-b-poly(MPC)
and PMPC-azo amphiphiles were determined by a dye solubi-
lization method using Nile Red (NR) as a probe molecule. NR in
THF (0.1 mg mLꢁ1, 30 mL) was added to a glass vial using a
microsyringe. Aer THF was evaporated, a micellar solution
(2 mL) was added. The concentration of the micellar solution
was varied from 0.1 to 5 ꢀ 10ꢁ4 mg mLꢁ1. Then, the solution
was stirred for 5 h. The uorescence measurements were taken
at an excitation wavelength of 550 nm and the emission was
monitored from 570 nm to 750 nm.
1H NMR (400 MHz, CDCl3): d ¼ 4.79–4.80 (d, 2H, CHCCH2-
CO), 4.71–4.74 (d, 2H, CH2OCO), 4.22–4.25 (d, 2H, CH2OCO),
2.54–2.55 (t, 1H, CHCCH2CO), 1.37 (s, 3H, CH3CC).
13C NMR (400 MHz, CDCl3): d ¼ 170.33, 147.22, 76.36, 75.95,
72.71, 53.47, 40.18, 17.39.
Typical procedure for the synthesis of PEG-b-poly(MPC)
The ring-opening polymerization of MPC was carried out under
an inert atmosphere of nitrogen using standard Schlenk-line
techniques. In a typical experiment, MPC (0.594 g, 3 mmol),
PEG5k (0.601 g, 0.12 mmol), TU (0.055 g, 0.15 mmol), DBU
Light-responsive release and re-encapsulation of NR
(0.005 g, 0.03 mmol) and dried DCM (10 mL) were placed in a NR was selected as a hydrophobic model drug to be encapsu-
dried Schlenk tube, tted with a rubber septum. The solution lated into the PMPC-azo micelles and uorescence spectroscopy
was further degassed by three freeze–pump–thaw cycles. The was used to investigate the release and re-encapsulation
resulting mixture was stirred at room temperature for 7 h, fol- behaviours of the micelles. NR in THF (0.1 mg mLꢁ1, 30 mL) was
lowed by precipitation in ice-cold diethyl ether and centrifuga- added to a glass vial via a microsyringe. Aer THF was evapo-
tion. The resulting product was collected by ltration and dried rated, a 0.5 mg mLꢁ1 micellar solution (4 mL) was added. Then,
under vacuum to yield a white powder. Yield: 1.087 g (91.0%).
the solution was stirred for 5 h. Aer the encapsulation process,
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 47929–47936 | 47931

View Article Online
RSC Advances
Paper
the uorescence spectrum of the micelles was immediately
recorded. The micelles were then exposed to 365 nm UV light for
15 min and subsequently irradiated with 450 nm visible light
for 80 min. Fluorescence measurements were performed at an
excitation wavelength of 550 nm and the emission was moni-
tored from 570 to 750 nm.
In vitro cytotoxicity assay
HeLa cells were used for studying the cytotoxicity of the
micelles. They were seeded in a 96-well plate at a density of 9.6
ꢀ 103 cells per well and incubated in DMEM at 37 ꢂC in 5% CO2
for 24 h. Then, the medium was removed and replaced with 200
mL polymer micelles. The aggregate concentrations of each
formulation were prepared by serial dilution with DMEM
medium. Aer treatment for 24 h, 20 mL of fresh medium
containing 10% of MTT of 5 mg mLꢁ1 stock was replaced in
each well. The plates were incubated for 4 h, and 230 mL of
DMSO was added to each well to dissolve intracellular MTT
formazan crystals, followed by absorbance measurement at 490
nm using a microplate reader (Varioskan Flash, Thermo
Scientic). Experiments were performed in triplicate.
Scheme 2 Synthesis of amphiphilic block copolymer PMPC-azo and
facile formation of reversible light-responsive micelles for drug
packaging and release.
Results and discussion
Synthesis and characterization of amphiphile PMPC-azo
The monomers of azo-N3 and MPC were synthesized according
to the design route shown in Scheme 1. The PMPC-azo
amphiphile was prepared via a two-step method (Scheme 2).
First, the PEG-b-poly(MPC) block copolymer was synthesized
through the ROP of MPC with PEG5k as a macroinitiator. The 1H
NMR spectrum of PEG-b-poly(MPC) in CDCl3 is shown in
Fig. 1C. The degree of polymerization (DP) of the polycarbonate
backbone was determined to be 24 by comparing the integrals
of peaks at d ¼ 3.36 (CH3O–, methyl protons of poly(ethylene
glycol) end group) with d ¼ 4.28 (–C(O)OCH2CCH2O–, methy-
lene protons of the carbonate units), which were close to the
theoretical value (Table 1). GPC analysis showed that the
obtained copolymer had a narrow polydispersity index (PDI) of
1.11 and a Mn of 10 544 g molꢁ1, which was in agreement with
1
those determined by H NMR end group analysis.
Second, the modication of PEG-b-poly(MPC) to prepare the
PMPC-azo amphiphile was performed in DMF with CuSO4/
sodium ascorbate as the catalytic system at room temperature
for 24 h via click reaction (Scheme 2). The crude copolymer was
puried by dialysis in order to remove the copper salt. Fig. 1
Fig. 1 1H NMR (in CDCl3) spectra of N3-azo (A), PMPC-azo (B) and
PEG-b-poly(MPC) (C).
compares the 1H NMR spectra of azo-N3, PMPC-azo and PEG-b-
poly(MPC). The signal at d ¼ 2.54 in Fig. 1C was assigned to the
protons of the alkynyl groups of PEG-b-poly(MPC), but this
signal disappeared in Fig. 1B aer click chemistry modication,
indicating that the propargyl groups of PEG-b-poly(MPC) have
been completely graed with azo-N3. Compared to Fig. 1A and
C, Fig. 1B revealed that signals assignable to azo-N3 were
detected at d 1.75–2.15, 6.90–7.34 and 7.87, while signals at d
1.22, 3.37, 3.65 and 4.26 owing to PEG-b-poly(MPC) were also
found in the PMPC-azo amphiphile. Moreover, the weak signal
at d 7.69, assigned to the proton of the triazole ring, evidenced
Scheme 1 Synthesis of azo-N3 and MPC.
47932 | RSC Adv., 2014, 4, 47929–47936
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Table 1 Molecular characteristics of amphiphiles PEG-b-poly(MPC)
and PMPC-azo
spontaneous self-assembly of micelles. PMPC-azo showed a
similar CMC value to PEG-b-poly(MPC) (0.0157 mg mLꢁ1 vs.
0.0145 mg mLꢁ1), suggesting that the self-assembled micelles
are thermodynamically stable in aqueous solution. These values
are consistent with literature data reported for gra copoly-
mers.24,35,41 Then, the size and morphology of the self-assembled
micelles were measured by DLS (Fig. 3A and B) and TEM (Fig. 4A
and B), respectively. From the TEM images, spherical
morphology was observed with an average size of 30 nm for
PEG-b-poly(MPC) micelles and an average size of 80 nm for
PMPC-azo micelles. Despite having a common spherical
morphology, PMPC-azo micelles have a larger DLS-determined
diameter than PEG-b-poly(MPC) micelles, which can be attrib-
uted to the expanded hydrophobic nucleus. These results
demonstrate that the PMPC-azo amphiphiles self-assembled in
aqueous solution into spherical micelles approximately 150 nm
in size, which contained an azo core stabilized by a PEG corona.
Entry
PEG-b-poly(MPC) 1.11
Mw/Mn
Mn,GPC
Mn,NMR
CMCc (mg mLꢁ1
)
a
a
b
10 544
16 285
9752
15 005
0.0145
0.0157
PMPC-azo
1.12
a
Both molecular weight (Mn,GPC) and the polydispersity (Mw/Mn) of the
amphiphiles were determined by GPC. b Mn,NMR was determined by 1H
NMR. c CMC: the critical micellar concentration of the amphiphiles was
determined by uorescence spectroscopy (Fig. 3).
the attachment of azo-N3 branches to the polyester backbone.
GPC results revealed that the PMPC-azo copolymer had a
narrow PDI of 1.12 and an Mn of 16 285 g molꢁ1, close to that
1
calculated by end group analysis from H NMR (Table 1). The
GPC trace of the gra copolymer showed a slight shi to lower
retention time, while maintaining narrow distributions with
dispersities similar to that of the unmodied copolymer PEG-b-
poly(MPC) (Fig. S6†). It is evident that PMPC-azo gra copol-
ymer can be readily prepared from propargyl-functionalized
polycarbonate via the copper catalyzed azide–alkyne cycloaddi-
tion (CuAAC) click reaction.
Photo-induced reversible self-assembly and disassembly
In order to investigate the photo-responsive self-assembly and
disassembly behaviors of the micelles, the PMPC-azo micelles
were examined by time-resolved UV-vis spectroscopy (Fig. 5).
DLS and TEM measurements of the samples were then con-
ducted and the images are shown in Fig. 3 and 4. Without
irradiation, the amphiphile PMPC-azo formed spherical
micelles with a diameter of approximately 150 nm in solution
(Fig. 3B) and approximately 80 nm in the dry state (Fig. 4B). The
diameters determined by TEM were smaller than those deter-
mined by DLS analysis, which was probably because of the
shrinkage of the PEG shell upon drying.38,42 Upon irradiation
with 365 nm UV light for 20 min, the diameter of the micelles
became smaller and the polydispersity was larger (Fig. 3C). At
the same time, the spherical micelles were disassembled as
shown in Fig. 4C. This phenomenon was because of the changes
in the hydrophilic-hydrophobic balance of PMPC-azo because
Self-assembling behavior of amphiphilic copolymers
The amphiphilic copolymers PEG-b-poly(MPC) and PMPC-azo
self-assembled into hydrophobic cored micelles stabilized with
hydrophilic PEG coronae (Scheme 2), and their self-assembly
behaviors were investigated in detail using uorescence spec-
troscopy, DLS and TEM. Using hydrophobic Nile Red as a probe,
uorescence spectroscopy can conveniently monitor the
micellar self-assembly and determine the critical micellar
concentration (CMC) of the amphiphiles.24,41 As shown in Fig. 2,
the emission uorescence intensity gradually increased with
increasing amphiphile concentration, suggesting the
Fig. 2 Fluorescence emission spectra of NR in PEG-b-poly(MPC) Fig. 3 Mean size distributions of the micelles determined by DLS:
micelles (A) and PMPC-azo micelles (C) of varying concentrations and PEG-b-poly(MPC) (A), PMPC-azo (B), PMPC-azo after 15 min of 365
the relevant emission intensity at 630 nm versus the log of concen- nm irradiation (C) and PMPC-azo after subsequent 80 min of 450 nm
tration for PEG-b-poly(MPC) micelles (B) and PMPC-azo micelles (D). irradiation (D).
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 47929–47936 | 47933

View Article Online
RSC Advances
Paper
the isomerization of trans-azobenzene into cis-azobenzene
under 365 nm UV irradiation.15,20,23 Moreover, UV-vis spectros-
copy was also employed to monitor this irradiation process
(Fig. 5A). The intensity of the characteristic absorption peak of
the p–p* transition of trans-azobenzene at around 340 nm
gradually decreased with increased irradiation time, and then
did not change. In addition, the absorption band at 440 nm,
which is ascribed to the n–p* transition of cis-azobenzene
became gradually stronger. These results suggested the
completion of isomerization of trans-azobenzene into cis-azo-
benzene within 15 min. As mentioned in the introduction, the
cis form of azobenzene is more polar than the trans form,
resulting in the destruction of the hydrophilic–hydrophobic
balance of PMPC-azo. As a result, the micelles disassembled.
Aer further irradiating the sample with visible light with a
wavelength of 450 nm for 80 min, the copolymer formed
micelles with an diameter of approximately 190 nm and a
polydispersity of 0.035 (Fig. 3D and 4D). This phenomenon
occurred because of the isomerization of cis-azobenzene into
trans-azobenzene. Fig. 5B showed the changes of the corre-
sponding UV-vis spectrum for the sample in the visible light
irradiation process. It can be seen that the absorption band at
around 340 nm, which corresponds to trans-azobenzene
showed a regular decrease in the n–p* absorption of cis-azo-
benzene suggesting the isomerization of cis-azobenzene into
trans-azobenzene.17,19,21 Because of the recovery of the hydro-
philic–hydrophobic balance of PMPC-azo, the micelles reas-
sembled aer 80 min of 450 nm irradiation. When comparing
Fig. 5A and B, aer irradiating with UV light and visible light,
the intensities of absorption at 340 nm and 450 nm were almost
identical to the original sample. These results indicated that the
PMPC micelles could undergo photo-induced reversible self-
assembly and disassembly. In order to further conrm that the
process is completely reversible, the PMPC-azo micelles were
irradiated with alternating UV and visible light (Fig. S7†). It can
be seen that the photo-isomerization of azobenzene groups in
PMPC-azo can occur reversibly many times. Aer three cycles,
the characteristic absorption peak of the p–p* transition of
trans-azobenzene at 340 nm was still the same as that of the
original micelles.
Fig.
4 TEM photographs of the micelles: PEG-b-poly(MPC) (A),
PMPC-azo (B), PMPC-azo after 15 min of 365 nm irradiation (C),
subsequent 80 min of 450 nm irradiation (D).
Model drug release and reload of NR-loaded micelles
Nile Red (NR) was used as a hydrophobic model drug to
demonstrate the concept of photo-triggered drug release
because the uorescence intensity of NR is known to substan-
tially increase in hydrophobic environments such as the interior
of micelles.42 Aer loading the NR, the uorescence spectrom-
etry of the samples was immediately carried out. As shown in
Fig. 6, the NR-loaded PMPC-azo micelles exhibited a photo-
triggered drug release prole. During the 15 min of 365 nm
irradiation, the uorescence emission intensity of NR gradually
decreased with the increase in irradiation time, indicating that
the model drug NR was released into water from the disinte-
gration of micelles (Fig. 6A).
Fig. 5 UV-vis spectra of the PMPC-azo micelles: (A) 365 nm UV light
irradiation induced trans-to-cis transition, and (B) 450 nm visible light
irradiation induced cis-to-trans transition.
By comparing uorescence emission intensities at 613 nm,
we found that 20% and 82% of drug molecules were released
47934 | RSC Adv., 2014, 4, 47929–47936
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 6 Fluorescence emission spectra of PMPC-azo micelles with
encapsulated NR after irradiation with: (A) 365 nm UV light for 15 min
and (B) subsequent 450 nm visible light for 80 min.
Fig. 7 Cell viability of the HeLa cell line against different concentra-
tions of micelles after being cultured for 48 h: (A) PEG-b-poly(MPC);
(B) PMPC-azo.
into water aer irradiation with UV light for 1 min and 15 min,
respectively. This phenomenon is attributed to the isomeriza-
tion of azobenzene under UV irradiation, which resulted in the
collapse of the micelles. Contrastingly, the characteristic peak
of NR became considerably stronger in Fig. 6B when the solu-
tion was irradiated by 450 nm visible light. Obviously, some
uorescent dye molecules were re-encapsulated into the
hydrophobic cores of the micelles. It is noted that the emission
cytotoxicity. This result can be mainly attributed to the excellent
biocompatibility of PEG and PC. It has been reported that
biocompatible nanocarriers with diameters of less than 200 nm
may avoid reticuloendothelial system (RES) recognition.1,2,4
Therefore, the micelles fabricated in this study have potential to
be an ideal drug carrier system.
peak did not increase further aer 80 min of irradiation, sug- Conclusions
gesting that not all NR molecules were encapsulated in the
In this paper, we present an azo-decorated block
process. The reason for the incomplete re-encapsulation might
be that the rate of micelle formation is higher than that of drug
loading.43 These results proved that the drug release and re-
encapsulation behaviors of the PMPC-azo micelles can be
controlled by irradiating with UV or visible light.
poly(carbonate) copolymer, PMPC-azo, via a convenient click
conjugation of photo-responsive azobenzene molecules to
propargyl-functionalized poly(carbonate)s. DLS and TEM
measurements revealed that the polymer can self-assemble into
spherical micelles with an average diameter of 150 nm in
aqueous solution. The CMC of the micelles was determined as
0.0157 mg mLꢁ1 by uorescence spectroscopy using Nile Red as
Cell cytotoxicity of PMPC-azo micelles
In order to test the cell cytotoxicity of PMPC-azo micelles, the a uorescence probe. Under alternative UV and visible light
MTT assay was used to evaluate the cytotoxicity of the micelles irradiation, the amphiphile PMPC-azo carried out a reversible
against HeLa cells aer 48 h of culture. As shown in Fig. 7, when micelle transition in water. Controlled release of model drug NR
the micelle concentration of PMPC-azo was 500 mg mLꢁ1, the under 365 nm UV light and re-encapsulation under 450 nm
cell viability still remained at about 95%, demonstrating the low visible light were conrmed by uorescence spectroscopy. The
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 47929–47936 | 47935

View Article Online
RSC Advances
Paper
reversibly light-responsive micelles with excellent biocompati- 20 S. Kadota, K. Aoki, S. Nagano and T. Seki, J. Am. Chem. Soc.,
bility, biodegradability and an appropriate size are highly 2005, 127, 8266–8267.
promising as smart carriers for controlled delivery of hydro- 21 F. D. Jochuma and P. Theato, Chem. Commun., 2010, 46,
phobic molecules for biomedical applications.
6717–6719.
22 B. Yu, X. Jiang, R. Wang and J. Yin, Macromolecules, 2010, 43,
10457–10465.
Acknowledgements
`
23 O. Boissiere, D. Han, L. Tremblay and Y. Zhao, So Matter,
Financial support from Planned Science and Technology
Project of Human Province (2012WK4017) and the Funda-
mental Research Funds for Central Universities are gratefully
acknowledged.
2011, 7, 9410–9415.
24 C. J. Chen, G. Y. Liu, X. S. Liu, D. D. Li and J. Ji, New J. Chem.,
2012, 36, 694–701.
˜
25 E. Blasco, J. L. Serrano, M. Pinol and L. Oriol,
Macromolecules, 2013, 46, 5951–5960.
26 W. Chen, H. Yang, R. Wang, R. Cheng, F. Meng, W. Wei and
Z. Zhong, Macromolecules, 2010, 43, 201–207.
Notes and references
1 N. Rapoport, Prog. Polym. Sci., 2007, 32, 962–990.
2 Y. Bae and K. Kataoka, Adv. Drug Delivery Rev., 2009, 61, 768–
784.
3 J. F. Lovell, T. W. Liu, J. Chen and G. Zheng, Chem. Rev., 2010,
110, 2839–2857.
27 W. Su, X. Luo, H. Wang, L. Li, J. Feng, X. Zhang and R. Zhuo,
Macromol. Rapid Commun., 2011, 32, 390–396.
28 J. A. Edward, M. K. Kiesewetter, H. Kim, J. C. A. Flanagan,
J. L. Hedrick and R. M. Waymouth, Biomacromolecules,
2012, 13, 2483–2489.
4 C. Oerlemans, W. Bult, M. Bos, G. Storm, J. F. W. Nijsen and
W. E. Hennink, Pharm. Res., 2010, 27, 2569–2589.
5 N. Kamaly, Z. Xiao, P. M. Valencia, A. F. Radovic-Moreno and
O. C. Farokhzad, Chem. Soc. Rev., 2012, 41, 2971–3010.
6 J. Z. Du, X. J. Du, C. Q. Mao and J. Wang, J. Am. Chem. Soc.,
2011, 133, 17560–17563.
7 L. Marta, M. G. G. Jose, Q. G. Isabel, G. Alberto and
P. Rodrigo, Macromolecules, 2011, 44, 3739–3745.
8 K. K. Venkata, S. F. Antoni, R. Janne and M. Raffaele,
Macromolecules, 2011, 44, 4569–4573.
29 J. Feng, R. X. Zhuo and X. Z. Zhang, Prog. Polym. Sci., 2012,
37, 211–236.
30 S. Tempelaar, L. Mespouille, O. Coulembier, P. Dubois and
A. P. Dove, Chem. Soc. Rev., 2013, 42, 1312–1336.
31 Y. Yamamoto, Y. Nagasaki, Y. Kato, Y. Sugiyama and
K. Kataoka, J. Controlled Release, 2001, 77, 27–38.
32 R. Duncan, Nat. Rev. Drug Discovery, 2003, 2, 347–360.
33 R. B. Greenwald, Y. H. Choe, J. McGuire and C. D. Conover,
Adv. Drug Delivery Rev., 2003, 55, 217–250.
34 P. F. Gou, W. P. Zhu and Z. Q. Shen, Biomacromolecules,
2010, 11, 934–943.
35 F. Coumes, V. Darcos, D. Domurado, S. Li and J. Coudane,
Polym. Chem., 2013, 4, 3705–3713.
9 Y. L. Cheng, C. L. He, C. S. Xiao, J. X. Ding, K. X. Ren, S. J. Yu,
X. L. Zhuang and X. S. Chen, Polym. Chem., 2013, 4, 3851–
3858.
10 N. Rapoport, Z. Gao and A. Kennedy, J. Natl. Cancer Inst.,
2007, 99, 1095–1106.
36 R. Yang, F. Meng, S. Ma, F. Huang, H. Liu and Z. Zhong,
Biomacromolecules, 2011, 12, 3047–3055.
11 Y. Wang, S. Gao, W. H. Ye, H. S. Yoon and Y. Y. Yang, Nat.
Mater., 2006, 5, 791–796.
37 V. Truong, I. Blakey and A. K. Whittaker, Biomacromolecules,
2012, 13, 4012–4021.
12 Q. Yan and Y. Zhao, J. Am. Chem. Soc., 2013, 135, 16300–
16303.
38 W. Chen, F. Meng, F. Li, S. J. Ji and Z. Zhong,
Biomacromolecules, 2009, 10, 1727–1735.
13 G. D. G. Bruno, M. J. Alain, D. Joseph and C. D. S. Stefaan,
Langmuir, 2006, 22, 5070–5074.
14 C. Wang, Q. S. Chen, Z. Q. Wang and X. Zhang, Angew.
Chem., Int. Ed., 2010, 49, 8612–8615.
15 J. M. Schumers, C. A. Fustin and J. F. Gohy, Macromol. Rapid
Commun., 2010, 31, 1588–1607.
16 Y. Zhao, Macromolecules, 2012, 45, 3647–3657.
17 A. Goulet-Hanssens and C. J. Barrett, J. Polym. Sci., Part A:
Polym. Chem., 2013, 51, 3058–3070.
39 W. Chen, Y. Zou, J. Jia, F. Meng, R. Cheng, C. Deng, J. Feijen
and Z. Zhong, Macromolecules, 2013, 46, 699–707.
40 W. Chen, Y. Zou, F. Meng, R. Cheng, C. Deng, J. Feijen and
Z. Zhong, Biomacromolecules, 2014, 15, 900–907.
41 R. C. Pratt, B. G. G. Lohmeijer, D. A. Long, P. N. P. Lundberg,
A. P. Dove, H. Li, C. G. Wade, R. M. Waymouth and
J. L. Hedrick, Macromolecules, 2006, 39, 7863–7871.
42 L. Sun, B. Zhu, Y. Su and C. M. Dong, Polym. Chem., 2014, 5,
1605–1613.
18 S. Swaminathan, J. Garcia-Amoros, A. Fraix, N. Kandoth,
S. Sortino and F. M. Raymo, Chem. Soc. Rev., 2014, 43,
4167–4178.
43 C. J. Chen, Q. Jin, G. Y. Liu, D. D. Li, J. L. Wang and J. Ji,
Polymer, 2012, 53, 3695–3703.
19 G. Wang, X. Tong and Y. Zhao, Macromolecules, 2004, 37,
8911–8917.
47936 | RSC Adv., 2014, 4, 47929–47936
This journal is © The Royal Society of Chemistry 2014