Novel azobenzene-based amphiphilic copolymers: Synthesis, self-assembly behavior and multiple-stimuli-responsive properties

Article abstract of DOI:10.1039/c8ra01660g

A series of novel azobenzene-based amphiphilic random copolymers P(POSSMA-co-AZOMA-co-DMAEMA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. A light and reduction dual-responsive azo group, pH-responsive terti

Full text of DOI:10.1039/c8ra01660g

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Novel azobenzene-based amphiphilic copolymers:
synthesis, self-assembly behavior and multiple-
stimuli-responsive properties†
Cite this: RSC Adv., 2018, 8, 16103
*
Yiting Xu,
Birong Zeng
Jie Cao, Qi Li, Jilu Li, Kaiwei He, Tong Shen, Xinyu Liu, Conghui Yuan,
and Lizong Dai
A
series of novel azobenzene-based amphiphilic random copolymers P(POSSMA-co-AZOMA-co-
DMAEMA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. A
light and reduction dual-responsive azo group, pH-responsive tertiary amine group and super
hydrophobic POSS moiety were incorporated into the polymer chain to generate multi-stimuli-
responsiveness. Self-assembly of these amphiphilic copolymers led to the formation of spherical
micelles in aqueous solution. The light, pH and reduction responsive properties of the micelles were
investigated systematically by DLS, TEM, UV-vis, FTIR and NMR. The azo groups can undergo trans–cis
isomerization under UV light irradiation, thus causing a diameter change of the micelles. Owing to the
large proportion of tertiary amine groups in amphiphiles, these micelles showed sensitive pH-response
behavior. The hydrophobic azo pendant in the polymer chain completely reduced to a more hydrophilic
substituted aniline in a reductive environment, resulting in the increase of overall hydrophilicity of
amphiphiles and the disassembly of polymeric micelles. Owing to these multi-stimuli–responses, the
polymeric micelles showed rapid and efficient release properties of hydrophobic molecules in response
to pH and reductive stimuli.
Received 26th February 2018
Accepted 25th April 2018
DOI: 10.1039/c8ra01660g
rsc.li/rsc-advances
reversible self-assembly and disassembly behaviors in aqueous
conditions by alternating UV and visible light irradiation. Hu23
1. Introduction
et al. synthesized a block copolymerpoly(ethylene glycol)-
modied poly(carbonate)s (PEG-b-poly(MPC)) and found its
reversible assembly–disassembly behavior in aqueous solution
triggered by visible light and UV light irradiation. Recently, an
azobenzene linkage cleavable by the enzyme azoreductase or
other reductants also has been reported. Rao and Khan
prepared an enzyme sensitive copolymer, which can be dis-
assembled through the cleavage of the azobenzene linkage in
copolymer chain in the presence of enzyme azoreductase.24 Li
and coworkers prepared prodrugs of 5-aminosalicylic acid
based on azobenzene molecule, and drug release can be trig-
gered by the cleavage of azobenzene with the reduction of
sodium hydrosulte.25
Stimuli-responsive polymeric systems integrating with special
responsive moieties can undergo alteration of their chemical
structures and physical properties in response to an external
stimulus, such as temperature,1,2 light,3,4 pH5–7 or reduction.8,9
Azobenzene and its derivatives can undergo trans–cis isomeri-
zation under UV or vis light irradiation. Also, the azo bond is
cleavable in a reducing medium, such as hydrazine (NH2NH2),
sodium hydrosulte (Na2S2O4) or azoreductase.10–12 Therefore,
the self-assembly behavior and the stimuli-responsive proper-
ties of azobenzene based copolymers, as well as their applica-
tions, were intensively investigated in recent years.13–17
Molecules containing azobenzene moieties have the ability to
form an ordered phase-separation structure in bulk, micelle,
vesicles, etc. Unique photoresponsive properties related with
the self-assembled structures have been observed and explored
for potential applications in areas such as optical devices,
sensors, and drug delivery.18–22 Photo-responsive micelles based
on azobenzene-modied amphiphilic copolymers showed
Polyhedral oligomeric silsesquioxane (POSS), the well
dened organic–inorganic hybrid compounds with inorganic
cubic core and outer organic groups,26,27 is a strong hydrophobic
hybrid molecule with nanoscale dimension. POSS has attracted
considerable attentions in building amphiphilic polymers due
to their unique merits including nontoxic, biocompatible and
chemically inert.28–31 A lot of POSS-containing amphiphilic
polymers have been designed and well-dened nanoaggregates
have been prepared through the hydrophobic aggregation of
Fujian Provincial Key Laboratory of Fire Retardant Materials, Xiamen University,
Xiamen 361005, People's Republic of China. E-mail: xyting@xmu.edu.cn; Tel: +86
18750256597
POSS segments in aqueous solution.32,33 For example, Matejka
ˇ
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra01660g
and coworkers built hybrid random copolymers containing
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POSS moiety via RAFT polymerization and these amphiphilic to 0 ꢀC, and NaNO2 (1.8975 g, 27.5 mmol) in water (20 mL) was
polymers can self-assemble into spherical micelles with PMA- added slowly. The mixture was stirred for 15 min in an ice bath
POSS segment as the hydrophobic part.34 In this contribution, to obtain a solution of diazonium salt, AZO-N2+, which was then
azobenzene monomer (AZOMA) was synthesized and used to added to a solution of 2-(N-ethylanilino)ethanol (4.1308 g, 25
prepare
a series of novel azobenzene-based amphiphilic mmol) in acetic acid aqueous solution (30 mL, 30 vol%). Aer
random copolymers via RAFT polymerization with the como- 5 min, K2CO3 aqueous solution (10 wt%) was added to adjust
nomers of methacrylate isobutyl POSS (POSSMA) and 2-(dime- the pH value to 7. Aer ltration, the resulting solid was puri-
thylamino) ethyl methacrylate (DMAEMA). The synthesis routes ed by column chromatography (gel silica gel, n-hexane : ethyl
of copolymer are described in Scheme 1. The incorporation of acetate, 1 : 1). Finally, the product was dried in a vacuum at
hydrophobic AZOMA and POSSMA units make it possible to room temperature for 12 h to afford 4.95 g (74%) of product.
self-assembly and form well-dened micelles. The hydro- The structure of HOAZO was characterized by FTIR and 1H NMR
1
phiphilic and pH-responsive DMAEMA units can stabilize the and ESI-MS spectroscopies. H NMR (400 MHz d6-DMSO, d in
micelles. Moreover, the introduction of azobenzene moiety and ppm) in Fig. S1:† 7.77 (4H), 7.52 (2H), 7.42 (1H), 6.83 (2H), 4.83
tertiary amine groups into polymer chains can endow these (1H), 3.58 (2H), 3.50 (4H), 1.14 (3H). The molecular weight of
amphiphilic copolymers with pH-, light- and reduction- 269 (C16H19N3O) is consistent with the molecular ion peak [M +
responsive properties. Based on the multi-stimuli- H]+ in Fig. S2.†
responsiveness of polymeric micelles, Nile red was selected as
a hydrophobic molecular model to study the guest molecular HOAZO (2.6934 g, 10 mmol) and triethylamine (2.0238 g, 20
A solution of 40-[2-(hydroxyethyl)ethylamino] azobenzene
encapsulation and release abilities of copolymers.
mmol) were dissolved in dichloromethane (100 mL). A solution
of methacryloyl chloride (1.5680 g, 15 mmol) in dichloro-
methane (15 mL) was added slowly to the above mixture in an
ice bath. The resultant mixture was stirred for 24 h at ambient
temperature. The solution was washed with the saturated
solution of NaCl, and the organic phase was dried over anhy-
drous MgSO4. The solvent was removed by rotary evaporation
and the residue was puried by column chromatography (gel
silica gel, n-hexane : ethyl acetate, 1 : 5). Finally, the product
was dried in a vacuum at room temperature for 12 h. A yield of
2. Experimental
2.1 Materials
Methacrylate isobutyl POSS (POSSMA) was obtained from
Hybrid Plastic Co. (USA) and used without further purication.
2-(Dimethylamino)ethyl
methacrylate
(DMAEMA)
was
purchased from Aladdin Chemical Co. Ltd. (Shanghai, China).
N-Ethyl-N-hydroxyethylaniline (96%) and the RAFT agent
cumyldithiobenzoate (CDB) was purchased from J&K Chemical
Ltd. (Shanghai, China). Aniline and sodium dithionite were
purchased from Sinopharm Chemical Reagent Co. Ltd.
(Shanghai, China). Azobisisobutyronitrile (AIBN) was recrystal-
lized from ethanol. All reagents in analytical grade were used as
received, unless otherwise have been noted before.
1
82% was obtained. H NMR (d1-CDCl3, d in ppm) in Fig. S3:†
7.86 (4H), 7.47 (2H), 7.38 (1H), 6.80 (2H), 6.11 (1H), 5.58 (1H),
4.36 (2H), 3.70 (2H), 3.52 (2H), 1.95 (3H), 1.24 (3H). The result of
ESI-MS analysis for AZOMA is shown in Fig. S4.† The molecular
weight of 337 (C20H23N3O2) is consistent with the molecular ion
peak [M + H]+ in Fig. S4.†
2.2 Synthesis of azobenzene monomer (AZOMA)
2.3 Synthesis of azobenzene-based copolymers P(POSSMA-
co-AZOMA-co-DMAEMA)
The synthetic route of the monomer AZOMA was illustrated
literature, according to the procedure similar to the published Azobenzene-based
copolymer,
P(POSSMA-co-AZOMA-co-
method.35 A solution of aniline (2.3280 g, 25 mmol) in DMAEMA), were synthesized with monomers of AZOMA, POS-
concentrated HCl aqueous solution (60 mL, 20 vol%) was cooled SMA and DMAEMA via RAFT polymerization as shown in
Scheme 1 Synthesis of copolymer of P(POSSMA-co-AZOMA-co-DMAEMA).
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Scheme 1. The RAFT polymerization was followed: AZOMA
(0.6748 g, 2.0 mmol), POSSMA (0.4526 g, 0.48 mmol), DMAEMA
(0.6288 g, 4.0 mmol), CDB (0.0109 g, 0.04 mmol), and AIBN
(0.0013 g, 0.008 mmol) were added into the Schlenk tube with
3.0 mL of THF. The polymerization ampoule was degassed with
ve freeze–pump–thaw cycles. Then the Schlenk tube was lled
with argon and placed in an oil bath at 65 ꢀC for 48 h. The
reaction was stopped into liquid nitrogen. Then the product was
precipitated in excess hexane, and washed with hexane three
encapsulated molecule mass
polymeric micelle weight þ encapsulated molecule mass
IE% ¼
%
2.7 Characterization
1H NMR spectra were recorded on a Bruker AV400 MHz NMR
spectrometer using d1-CDCl3 and d6-DMSO as the solvent. FTIR
measurements were conducted on an AVATAR 360 FTIR (Nicolet
times. The resulting red powder was dried overnight under Instrument) at room temperature. Molecular weight determi-
vacuum at room temperature.
nations for monomers were conducted on an Esquire 3000 plus
FTIR (KBr, wavenumber in cmꢁ1) (Fig. S6†): 2953, 2821, 2770, mass spectrometer (Bruker Daltonics) and the solvent was
methanol at the concentration of 1 mg mLꢁ1. Molecular weight
determinations for polymers were made using GPC analyses
1728, 1600, 1513, 1462, 1399, 1135, 1017, 962, 822, 766, 746,
690. 1H NMR (d1-CDCl3, d in ppm) (Fig. S7†): 7.82 (s, 4H), 7.45 (s,
performed in THF using a series of waters styragel HR2, HR4,
and HR5. The eluent was THF at a ow rate of 1.0 mL minꢁ1. A
series of low polydispersity polystyrene standards were used for
the GPC calibration. Dynamic Light Scattering (DLS) measure-
ments were used to measure the zeta potential, aggregate size
and size distribution and performed on a Zetasizer Nano ZS
Instrument (Malvern Instruments, UK) at a scattering angle of
90ꢀ, and analyzed by Malvern Zetasizer soware version 6.20.
Transmission Electron Microscopy (TEM) analysis was per-
formed on a JEM2100 transmission electron microscope with
an accelerating voltage of 200 kV. One drop of micelles solution
was placed on a copper-mesh coated with carbon and then air-
dried before measurement. The UV-vis spectra of P(POSSMA-co-
AZOMA-co-DMAEMA) micellar solutions at various condition
were measured using a UV-2550 spectrometer (SHI-MADZU).
The uorescence emission spectra were measured using
a Horiba Fluoromax-4.
2H), 7.36 (s, 1H), 6.78 (s, 2H), 4.04 (s, 2H), 3.86 (m, 2H), 3.62 (s,
2H), 3.48 (s, 2H), 2.54 (s, 2H), 2.26 (s, 3H), 1.84 (d, 2H), 0.59 (d,
16H).
2.4 Synthesis copolymers of P(POSSMA-co-DMAEMA),
P(AZOMA-co-DMAEMA), P((POSSMA-co-AZOMA)-b-DMAEMA)
and P(POSSMA-co-AZOMA-co-DMAEMA) as control samples
As control samples, P(AZOMA-co-DMAEMA), P(POSSMA-co-
DMAEMA), P(POSSMA-co-AZOMA)-b-P(DMAEMA) were synthe-
sized by RAFT polymerization, and random copolymers of
P(POSSMA-co-AZOMA-co-DMAEMA) was prepared by conven-
tional free radical polymerization. Their synthetic process was
1
provided in ESI.† The copolymers were characterization by H
NMR spectra (Fig. S7†) and GPC (Fig. S8†).
2.5 Preparation of copolymer micelles in aqueous solution
P(POSSMA-co-AZOMA-co-DMAEMA) random copolymers and
control samples and were dissolved in THF. Then pre-
determined volume Milli-Q water, which is a selective solvent
for copolymers, was added into the solution at the speed of 1.0
mL minꢁ1, to induce the self-assembly of copolymer. Aer-
wards, the mixture was stirred for 3 days at ambient tempera-
ture to remove the THF.
3. Results and discussion
3.1 Synthesis of azobenzene-based copolymer
Azobenzene-based
copolymer,
P(POSSMA-co-AZOMA-co-
DMAEMA), AZOMA, POSSMA and DMAEMA via RAFT poly-
merization were shown in Scheme 1. Table 1 summarizes the
results obtained for copolymers with different ratio of the
monomers to CDB. Three types of P(POSSMA-co-AZOMA-co-
DMAEMA)s denoted as RCP-1, RCP-2 and RCP-3 were prepared,
the molecular weights of which are 1.375 ꢂ 104 (PDI ¼ 1.17),
1.973 ꢂ 104 (PDI ¼ 1.26), 2.175 ꢂ 104 (PDI ¼ 1.30) (Fig. S10†),
respectively. Control samples of P(AZOMA-co-DMAEMA),
2.6 Preparation and characterization of Nile red-loaded
micelles
P(POSSMA-co-AZOMA-co-DMAEMA) random copolymers and
Nile red were dissolved in THF. Then predetermined volume
Milli-Q water was added into above solution to induce the self-
assembly of P(POSSMA-co-AZOMA-co-DMAEMA) and load Nile
red molecule. Aerwards, the solution was dialysis against
Milli-Q water using a dialysis membrane with a molecular
weight cutoff of 3000 Da for 2 days. The standard solutions of
Nile red were also prepared to build standard work curve. The
incorporation efficiency (IE) and loading capacity (LC) were
calculated using below equations.
P(POSSMA-co-DMAEMA)
and
P(POSSMA-co-AZOMA)-b-
P(DMAEMA) were synthesized by RAFT polymerization denoted
as C-1, C-2, C-3, and P(POSSMA-co-AZOMA-co-DMAEMA) was
prepared by conventional free radical polymerization denoted
as C-4. Their experimental conditions and results were also
shown in Table 1.
FTIR and NMR are also applied to characterize the copolymer,
the results of which are shown in Fig. S6 and S7.† The absorption
peaks at around 2821 cmꢁ1 and 2770 cmꢁ1 are attributed to
–NCH3 and –NCH2– in DMAEMA units. Compared to AZOMA
monomer, we assign the broad absorption peak at 1115 cmꢁ1 to
overlapping peaks of C–N bond in AZOMA units and Si–O–Si
bond in POSSMA units. In Fig. S8,† there are no NMR signals in
encapsulated molecule mass
LC% ¼
%
total molecule mass
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Table 1 Experimental conditions and results of the RAFT or conventional free radical polymerization to obtain copolymers of P(POSSMA-co-
AZOMA-co-DMAEMA) or control samplesa
[POSSMA] : [AZOMA] :
[POSSMA] : [AZOMA] :
[DMAEMA]b
Sample
[DMAEMA] : [CDB] : [AIBN]a
Time (h)
10ꢁ3 Mn (GPC)
PDI
Polymer yield
RCP-1
RCP-2
RCP-3
C-1
12 : 50 : 100 : 1.25 : 0.25
12 : 50 : 100 : 1.00 : 0.20
12 : 50 : 100 : 0.75 : 0.15
0 : 50 : 100 : 1.00 : 0.20
12 : 0 : 100 : 1.00 : 0.20
12 : 50 : 100 : 1.00 : 0.20
12 : 50 : 100 : 0 : 1.00
0.17 : 1.0 : 1.9
0.16 : 1.0 : 2.1
0.18 : 1.0 : 2.2
0 : 1.0 : 2.27
1.0 : 0 : 7.76
0.18 : 1.0 : 2.93
0.21 : 1.0 : 2.56
48
48
48
48
48
48
48
11.78
15.55
16.66
11.4
9.43
18.6
1.17
1.26
1.30
1.27
1.22
1.34
2.32
63.2%
66.1%
59.4%
47.7%
56.2%
51.3%
41.3%
C-2
C-3c
C-4
19.3
a
The feed molar ratio of monomers, the chain transfer agent and initiator. b The composition ratio in copolymer chains determined from 1H-NMR
spectra by the integral areas of characteristic peak relevant to every structural unit. For example, the composition ratio of RCP-1 was calculated as
c
[POSSMA] : [AZOMA] : [DMAEMA] ¼ A0.59/16 : A7.82/4 : A2.26/6, in which A was areas for short. Block copolymer C-3 is synthesized by RAFT
polymerization using P(POSSMA-co-AZOMA) as macromolecular chain transfer agent with feed molar ratio of [P(POSSMA-co-
AZOMA)] : [DMAEMA] : [AIBN] ¼ 1 : 100 : 0.20, and P(POSSMA-co-AZOMA) macromolecular chain transfer agent is synthesized with feed molar
ratio of [POSSMA] : [AZOMA] : [CDB] : [AIBN] ¼ 12 : 50 : 1.00 : 0.20.
the d range of 5.5 to 6.5 ppm, which belongs to vinyl protons in shapes with a well-dened core–shell structure, in which
monomers, indicating that the purication by precipitation hydrophobic POSSMA and AZOMA is as cores and hydrophilic
removes the residual monomers. The signals of the aromatic ring DMAEMA as shells. The hydrophobic effect of POSS moieties
protons at 7.82 ppm for AZOMA units and the methyl protons in and p–p stacking effect of AZO moieties induce molecular
tertiary amine of DMAEMA units at 2.26 ppm and the methylene chain aggregate in water. The hydrogen bonds between
protons next to silicon in POSSMA units at 0.59 ppm demonstrate DMAEMA and water, and the protonated DMAEMA in weakly
that three monomers are introduced into the copolymer chains acidic Milli Q water stabilize the aggregates. And the zeta
as expected. The relative ratio of POSSMA, AZOMA and DMAEMA potential of the micellar solution is +5.92 mV. It is indicated
units in copolymer chains can be determined by calculating the that micelle surface charges are positive. The 1H NMR spectrum
integral ratio among the characteristic signal peaks in Fig. S11.† of micelles of random copolymer in D2O is shown in Fig. S12,†
The ratio of three azobenzene-based copolymer [POS- in which only special peaks for DMAEMA are displayed:
SMA] : [AZOMA] : [DMAEMA] is 0.17 : 1.0 : 1.9, 0.16 : 1.0 : 2.1 1.75 ppm for –N(CH3), 3.25 ppm for –N(CH2), 3.75 ppm for
and 0.18 : 1.0 : 2.2, respectively.
–O(CH2). It is further that the shell of micelles consists of
DMAEMA. The large hydrophobic core volume might be resul-
ted from the aggregation of POSS moieties in polymer side
chains, which have nature nanoscale at about 1.5 nm and neat
space structure. The characteristic structure makes micelles
more advantageous in encapsulating of hydrophobic drugs. The
DLS result in Fig. 1b indicates that the micelles have an average
diameter of 155.5 nm with a size distribution of PDI ¼ 0.156.
The difference between the sizes measured by DLS and deter-
mined by TEM can be attributed to the fact that the DLS
measures swollen micelles in aqueous solutions, and the sizes
observed by TEM are derived from the dried micelles. The
micellization process of the copolymer is shown in Fig. 1c.
In order to clarify the formation of the assembled structures
from amphiphilic random copolymer of P(POSSMA-co-AZOMA-
co-DMAEMA), some control samples self-assembly aggregates
are also studied. Their SEM photos are shown in Fig. 2. The C-1
can self-assemble in water to form spherical micelles with
hydrophobic AZOMA as cores and hydrophilic DMAEMA as
shells. But the aggregates of C-2 is irregular, which is because
the ratio of hydrophobic POSSMA moieties to too less than
AZOMA in C-1, and there is no appropriate hydrophilic–hydro-
phobic balance in C-2 to form spherical micelles. The micelles
of C-3 are uniform due to its narrow dispersity in both molec-
ular weight and block length. The C-4 also can self-assemble in
water to form spherical micelles with a broad dispersion of size
3.2 The morphology of P(POSSMA-co-AZOMA-co-DMAEMA)
self-assembly aggregates in aqueous solution
A typical TEM image of the micelles formed by RCP-2 with
a concentration of 2.5 mg mLꢁ1 (pH ¼ 6.9, 25 C) in aqueous
ꢀ
solution is presented in Fig. 1a. The micelles display spherical
Fig. 1 TEM image (a) and DLS result (b) of 2.5 mg mLꢁ1 RCP-2 micelles
(pH ¼ 6.9, 25 ꢀC) and schematic representation for the self-assembly
process of P(POSSMA-co-AZOMA-co-DMAEMA) (c).
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Fig. 2 TEM images of control samples, (a) P(AZOMA-DMAEMA), (b) P(POSSMA-co-DMAEMA),(c) P(POSSMA-co-AZOMA)-b-P(DMAEMA) by
RAFT, (d) P(POSSMA-co-AZOMA-co-DMAEMA) by conventional free radical polymerization.
distribution. It is because that molecular weight of C-4 via
conventional free radical polymerization has a broader poly-
dispersity than that of controlled RAFT polymerization.
According to the self-assembly behaviors of control samples, we
know that amphiphilic random copolymers can form micelles,
and there are many factor inuence its morphology. Copolymer
composition is an important factor in the process of self-
assembly of random copolymers. Self-assembly of random
copolymers is largely dependent on their hydrophilic–hydro-
phobic balance (oen referred to as the hydrophilic–lipophilic
balance (HLB)).36
The initial concentration of amphiphilic copolymer is one of
important factors in the self-assembly process, which inu-
ences the morphology and size of self-assembled aggregates.
The RCP-2 micelles at different initial concentration. As shown
in Fig. 2, the diameter of micelles with 1.0 mg mLꢁ1, 2.5 mg
mLꢁ1 and 5.0 mg mLꢁ1 is 121.1 nm (PDI ¼ 0.160), 155.5 nm
(PDI ¼ 0.156) and 196.4 nm (PDI ¼ 0.178), respectively. This
increase of micellar diameter induced by initial concentration is
also observed in the TEM images in Fig. 3. Furthermore, the
increase of initial concentration results in the increase the
number of micelles and deformation of micelles, but does not
change the core–shell structure of micelle.
3.3 Light-responsive properties of P(POSSMA-co-AZOMA-co-
DMAEMA) micelles
Fig. 3a shows the hydrodynamic radius of RCP-2 micelles at
1.0 mg mLꢁ1 upon light irradiation at 365 nm. Aer UV light
irradiation, the sizes of micelles decrease in the rst 5 min, and
then remain unchanged. It can be explained from the trans–cis
isomerization of the azo moieties, which results in the decrease
of molecular size and form a more compact aggregate in the
core of micelles. The UV-vis absorption spectra before and aer
the irradiation of UV light are shown in Fig. 4b. The absorption
of p–p* transition band of typical aminoazobenzenes at 414 nm
reduced rapidly aer 1 min of UV light irradiation, and
substantially retained the same value. It indicates the isomeri-
zation of azo groups from trans conguration to cis congura-
tion and the photoisomerization reaches a balance state in
a short time. Due to the transition band of p–p* and n–p* is
close, the n–p* might be completely buried beneath the intense
p–p*, one could not identify the absorption of azo-group in cis
conguration.
3.4 pH-responsive properties of P(POSSMA-co-AZOMA-co-
DMAEMA) micelles
We also prepared micellar solution of RCP-1, RCP-2 and
RCP-3 at the concentration of 2.5 mg mLꢁ1, whose sizes and size
distributions are shown in Fig. 3. In the case of RCP-1, the size
of micelles is 106.0 nm. And the size increase to 155.5 nm and
176.4 nm for RCP-2 and RCP-3, respectively. TEM images
conrm this trend that the micelle size increased as Mw
increasing. This result derived from that the more hydrophobic
molecular with higher Mw, a larger micelles in size should be
formed to achieve hydrophobic balance.
The critical micelle concentrations (CMC) of RCP-1, RCP-2
and RCP-3 are also measured by a uorescence method with
hydrophobic Nile red as a uorescent probe. Fluorescence
spectroscopy (lex ¼ 560 nm) is utilized to monitor the self-
assembly process. The CMC of RCP-1, RCP-2 and RCP-3 is
found to been 0.258 mg mLꢁ1, 0.239 mg mLꢁ1 and 0.181 mg
mLꢁ1, respectively. Despite the three units ratio is pretty similar
in RCP-1, RCP-2 and RCP-3, the increase in molecular weight
will improve the hydrophobicity of the whole polymer chains.
Furthermore, the longer polymer chain length possesses the
stronger intermolecular interaction. These factors can increase
the driving force of self-assembly process, which means that
this amphiphilic copolymer can form micelles at a lower
concentration.
There are subtle differences in the acid–base properties of
human tissues, for example, the pH value ranges from 1 to 2 in
the stomach, 8 to 9 in the small intestine, and the pH value
drops to 5.8–7.2 in tumor tissues due to the production of lactic
acid. According to this difference in pH, pH-responsive assem-
blies and drug delivery systems can be designed to achieve site-
specic drug release and enhance the utilization of drugs by
control pH value. Incorporation of pH-responsive DMAEMA
units into a polymer chains endows the polymeric micelles with
pH-response property. The pH dependence of the aggregates for
RCP-2 solution (2.5 mg mLꢁ1) is examined by DLS and TEM.
The results are revealed in Fig. 5c and d, the changes of
aggregates can undergo two stages with the variation of pH. The
micellar size remarkably increases from 154.4 to 216.5 nm when
pH decreases from 6.9 to 6.0. As shown in Fig. 5a and b, the
clear shell–core spherical structure can be identied when pH
value is 6.5. However, when pH value further decreases to 6.0,
the spherical structure of micelle has been destroyed and
formed irregular polymer aggregates. When pH is further
decreased to 5.5, the size distribution of aggregate is much
broader (PDI ¼ 0.475) and the main peak is about 10–15 nm,
which indicates that polymeric micelles have been dis-
assembled. This increase in aggregate size in the pH range of
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Fig. 3 TEM images (a)–(c) and DLS results (a0)–(c0) of RCP-1, RCP-2 and RCP-3, TEM images (d)–(f) and DLS results (d0)–(f0) of RCP-2 micelles at
various initial concentration of 1.0 mg mLꢁ1, 2.5 mg mLꢁ1 and 5.0 mg mLꢁ1
.
6.9–6.0 can be ascribed to the higher degree of protonation and DMAEMA) copolymer with a large proportion of protonated
molecular repulsion of the DMAEMA unites. The decrease in DMAEMA units (about 63.29 mol%). Further, the tertiary amino
aggregate size at pH 5.5 may derive from the disruption of groups in AZOMA units can also be protonated in an acidic
hydrophilic–hydrophobic balance of P(POSSMA-co-AZOMA-co- solution. These structural factors endow P(POSSMA-co-AZOMA-
Fig. 4 The change in size of RCP-2 micelles upon irradiation of UV light (a) and the change in their UV-vis spectra (b).
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Fig. 5 TEM images of micelles at various pH: (a) pH ¼ 6.5, (b) pH ¼ 6.0, and diameters (c) and trend (d) of self-assembly micelles at various pH.
co-DMAEMA) micelles with sensitive pH-response property, the UV-vis absorption intensity of P(POSSMA-co-AZOMA-co-
which slightly changes in pH value can induce signicant DMAEMA) micelles upon the addition of reductant Na2S2O4. As
changes in the polymer's aggregates. It also can be found that shown in Fig. 6a, with the addition of Na2S2O4, the absorption
the aggregates are deformed and the core–shell structure of trans azobenzene (416 nm) decreases signicantly and
disappear in the TEM photos in Fig. 7. There are even a large eventually disappears, which indicates that azo pendant in
number of residual irregular polymer aggregates for the disas- polymer chain completely cleavages. For this reason, the yellow
sembly of micelles when pH is 6.0. Fig. 7c describes the pH- micellar solution turns colorless (Fig. 6a). DLS data shows that
induced micellization behavior of P(POSSMA-co-AZOMA-co- micelle particle size increased to 189.9 nm upon adding
DMAEMA).
Na2S2O4 aqueous solution. The spherical micelles with core–
shell structure were completely deformed to irregular nano-
aggregates (Fig. 6b and c), indicating that polymer micelles
have been disassembled. This micelle morphology transition
can be attributed to the fact that azo pendant in polymer chain
completely reduced to the corresponding cleaved product aer
adding 10 mg Na2S2O4 (Fig. 7a). The cleaved product, namely
substituted aniline, shows more hydrophilic than azopendant
3.5 Reduction-responsive property of micelles
Azobenzene moiety is a dual-responsive moiety to ultraviolet
light irradiation and reductive environment. Sodium dithionite
(Na2S2O4) is selected as reductant to investigate the reduction-
responsive property of P(POSSMA-co-AZOMA-co-DMAEMA)
micelles. UV-vis spectroscopy is used to monitor the change of
Fig. 6 UV-vis spectra and photograph of RCP-2 micelles solution upon reduction of Na2S2O4 and the inset of (a) represents the photograph of
micelles solution, and their TEM images (b and c) and the inset of (b) represents DLS date, (d) simulate images of RCP-2 micelles solution upon
reduction of Na2S2O4.
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Fig. 7 Reduction reaction of P(POSSMA-co-AZOMA-co-DMAEMA) (a) and the FTIR spectra of RCP-2 and product after reduction of Na2S2O4 (b)
and the 1H NMR of product after reduction of Na2S2O4 (c).
and the proportion of AZOMA units in copolymer is about (d6-DMSO) is shown in Fig. 7c. The signals of protons in –SiH2–
31.65 mol%, resulting the disruption of hydrophilic–hydro- of POSSMA units at 0.58 ppm (16H), the protons of in –NCH3 of
phobic balance of P(POSSMA-co-AZOMA-co-DMAEMA) copol- DMAEMA units at 2.78 ppm (6H) and the peaks at d ¼ 6.66 ppm
ymer in solution. Due to the formation of hydrogen bonds (4H) correspond to the protons of the benzene ring for
between the substituted aniline and water and the strong substituted aniline. The signals of the protons of in benzene
hydrophobic POSSMA units (about 5.06 mol%), the polymeric units shi from 7.82, 7.36 to 6.7 ppm, and their integral area
micelles deformed into irregular nano-aggregates. This analysis ratio decrease in comparison with RCP-2. By integrating these
is based on the reduction reaction of P(POSSMA-co-AZOMA-co- three
peaks,
it
can
be
calculated
that
[POS-
DMAEMA) copolymer. So, it is necessary to analyze the cleaved SMA] : [AZOMA] : [DMAEMA] ratio is 0.17 : 1.0 : 2.2, which is
product of copolymer. Na2S2O4 is selected as reductive agent to similar to and the ratio of 0.16 : 1.0 : 2.1 for the copolymer
get the cleaved product of copolymer and 1H NMR and FT-IR are before reduction. Accordingly to these structural analysis to the
used to analysis the structure of the product for micelle solution reduced product, we conrm that the azobenzene-based
treated by Na2S2O4. The FTIR spectrum of RCP-2 has been amphiphiles can be cleaved in azobenzene moiety and this
changed signicantly aer reduction (Fig. 7b). Owing to the reduction process will not affect the main chain of polymer.
cleavage of azo bond, half of benzene ring in azobenzene moiety Since the hydrophobic azobenzene side strand breaks to form
is separated from the end of the side chain. As a result, the a more hydrophilic side groups of substituted aniline, the
overlapping peaks at about 1600 cmꢁ1 is signicantly weaken, copolymer transforms to a more hydrophilic macromolecule
which attributes to the benzene ring skeleton vibration and the and induces the disassemble of micelles.
azo bond vibrations. At the same time, the absorption of C–N
bond at about 1399 cmꢁ1 is signicant reduced. In addition,
3.6 Control release property of micelles
C–H deformation vibration on the phenyl ring characteristic
Multi-responsive polymeric micelles with core–shell structure is
peak have also been signicantly changed. Aer the reduction
a good candidate for encapsulating and releasing hydrophobic
of 1,4-substituted benzene ring the characteristic peaks at
guest molecules, in which the hydrophobic core can provide
823 cmꢁ1, 770 cmꢁ1 and 689 cmꢁ1 for mono-substituted
a large space for encapsulating guest molecules and the
benzene ring disappear. However, C–H characteristic absorp-
hydrophilic shell can provide protect the encapsulated mole-
tion of 1,4-substituted benzene peak at 749 cmꢁ1 still exists.
cules. What's more, the encapsulated molecules can be released
We can clearly distinguish the signals of three structures
upon the changes of environment. Given P(POSSMA-co-AZOMA-
units in the reduced product of RCP-2, whose 1H NMR spectrum
co-DMAEMA) micelles have a greater volume of the hydrophobic
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(Fig. S14†) and the equation was y ¼ 1185.25 + 5.91 ꢂ 107x. The
results are shown in Table 2.
Table 2 IE and LC of Nile red in copolymer micellar solution (1.0 mg
mLꢁ1
)
Among current responsive drug delivery systems, pH is one
of the most popular stimuli and has been explored extensively.
As shown in Fig. 8(a)–(c), the uorescence intensity of Nile red
encapsulated in micelles signicantly reduced in the function
of time in acidic environment. By comparing uorescence
emission intensities at 620 nm in Fig. 7d, we found that the
relative uorescence intensity decreased to 80.01%, 58.73% and
32.21% within 20 min in pH 6.5, 6.0 and 5.5, respectively. In
Sample
IE (%)
LC (%)
RCP-1
RCP-2
RCP-3
31.25
36.42
40.73
23.1
28.01
33.25
core and a good pH and reducing response characteristics, the addition, the relative intensity decreased to 67.70%, 51.37%
encapsulating and release properties of micelles are discussed. and 26.82% within 140 min in various pH environment. These
Nile red was selected as a hydrophobic molecular model to results indicate that the acid environment can induce the effi-
study the properties of encapsulating and releasing hydro- cient release hydrophobic model molecule encapsulated in
phobic molecules in response to the pH and reductive stimulus. micelles. And the release rate and efficiency increase as the
The copolymer micelles encapsulated Nile red were prepared, IE acidity increases. This phenomenon is attributed to the sensi-
and LC of Nile red in copolymer micelles was determined by tive pH-responsive property of polymeric micelles.
uorescence spectroscopy with excitation wavelength of 490 nm
The reduction-responsive property of polymeric micelle
and the emission intensity at 620 nm. The standard work curve endows it steady release the loaded hydrophobic model mole-
is built with correlation coefficient (R2) value at least 0.999 cule. As shown in Fig. 9, the relative uorescence intensity at
Fig. 8 Fluorescence emission spectra of Nile red encapsulated micelle solutions (a)–(c) and the relationship between I620 nm and time (d) on
treatment with various pH.
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Fig. 9 Fluorescence emission spectra of Nile red encapsulated micelle solutions (a) and the relationship between I620
and time (b) on
nm
treatment with Na2S2O4 aqueous solution.
620 nm of Nile red decreases to 17.1% within 20 min and 12.5%
within 39 min, demonstrating that the most of model molecule
Conflicts of interest
encapsulated in micelles can be released into water aer adding
Na2S2O4. This steady release property can be explained by the
disassembly of micelles in a reductive environment.
There are no conicts to declare.
Acknowledgements
The authors acknowledge the National Natural Science Foun-
dation of China (51273164, 51573150) for funding. Financial
support from Scientic, Xiamen Science and Technology Major
Project (3502Z20171002), and Technological Innovation Plat-
form of Fujian Province (2014H2006) is also gratefully
acknowledged.
4. Conclusions
In this paper, we synthesized a series of azobenzene-based
copolymers, P(POSSMA-co-AZOMA-co-DMAEMA) via RAFT
polymerization. In this way, the light and reduction dual-
responsive azo group, pH-responsive tertiary amine group
and super hydrophobic POSS moiety were incorporated into
the polymer chain to generate multi-stimuli-responsive
amphiphiles. The effects of initial concentration and molec-
ular weight on micellar morphology and size were investigated
by TEM and DLS and revealed that the sizes of micelles tend to
increase as the concentration or molecular weight increases.
The azo groups can undergo trans–cis isomerization under UV
light irradiation and the diameters of micelles decreased.
Owing to the large proportion of tertiary amine groups in
amphiphiles, the micelles showed sensitive pH-responsive
property. Hydrophobic azo pendant in polymer chain
completely reduced to the corresponding more hydrophilic
substituted anilines in a reductive environment, resulting in
the increase of overall hydrophilicity of amphiphiles and the
disassembly of polymeric micelles. Based on the pH- and
reductive-stimuli-responses of micelles, we explored the
release properties of self-assembly micelles by uorescence
spectroscopy. As expected, the acid or reducing environment
can induce the rapid and efficient release of hydrophobic
molecules loaded in micelles. Hence, as a novel azobenzene-
based amphiphiles with distinctive virtues, these multi-
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