Polymer

Chemistry
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Copper-coordination induced fabrication of

Cite this: Polym. Chem., 2021, 12,
stimuli-responsive polymersomes from
3105 amphiphilic block copolymer containing pendant
thioethers†
Shuqi Dong,a Li Liu *a and Hanying Zhao a,b

In this work, we synthesized oxidation-responsive amphiphilic block copolymers PEG45-b-P(MET/PBC)n

Received 18th March 2021,
Accepted 30th April 2021
DOI: 10.1039/d1py00371b
rsc.li/polymers
bearing pendant phenylboronic ester carbamate (PBC) and thioether moieties in the hydrophobic block
by RAFT polymerization and post-polymerization modification. As the hydrophobic block length
increased, the polymeric self-assemblies underwent a morphological transition from spherical micelles to
worm-like micelles to bilayered polymersomes. Triggered by H2O2, the polymersomes disintegrated
because of the oxidation of thioether to sulfoxides and the decomposition of PBC moieties. Taking
advantage of the thioether–copper coordination capability, the hybrid polymersomes with Cu2+-cross-
linked membrane were fabricated via the co-assembly of the block copolymer with Cu2+ ions, driven by
the coordination interactions between the hydrophobic block and Cu2+ ions. The metal–ligand inter-
action endows the hybrid polymersomes with a responsive property to the competitive ligand. In the
presence of glutathione (GSH) (or sodium ascorbate) and H2O2, Cu+ ions were in situ produced via the
reduction of the entrapped Cu2+ ions and subsequently initiated a Fenton-like reaction to generate
hydroxyl radicals. The catalytic activity of the hybrid polymersomes-mediated Fenton-like reaction was
evaluated by the oxidation of terephthalic acid and the degradation of methylene blue, respectively. In the
presence of H2O2 and GSH, the hybrid polymersomes underwent a shape change and transformed into a
mixture of spherical micelles and worm-like micelles.

Introduction have attracted considerable interest because of the facile gene-

Polymersomes, analogous to liposomes from lipids, are
enclosed membrane structures made from amphiphilic
polymers.1,2 Over the last two decades, great advances have
been achieved in the fabrication of intelligent polymersomes
from stimuli-responsive block copolymers, which have diverse
applications in drug delivery, nanoreactors, and protocells.3–9 In
the presence of environmental stimuli such as pH, temperature,
reduction, oxidation, light, and shear stress, the responsive
polymersomes can undergo disassembly or structural reorganiz-
ation. Among these stimuli, the reactive oxygen species (ROS)
a In recent years, there has been an emerging interest in
Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of
Polymer Chemistry, College of Chemistry, Nankai University, P. R. China.
E-mail: nkliuli@nankai.edu.cn
b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300071, P. R. China
† Electronic supplementary information (ESI) available: 1H NMR spectrum of C1,
GPC traces, TEM image of PEG45-b-P(MET/PBC)15/Cu2+ co-assemblies, DLS of
HP1–3, SEM image and elemental mapping analysis and Ellman’s assay. See
DOI: 10.1039/d1py00371b
ration of ROS with spatiotemporal control by in situ biochemical
or photochemical reactions. For example, glucose oxidase-cata-
lyzed oxidation of glucose can produce H2O2 to induce the
destabilization of oxidation-sensitive polymersomes.10 So far, a
diversity of polymeric materials, containing thioether,11,12 sel-
enium/tellurium,13 thioketal,14 phenylboronic ester functional-
ities,15 etc., have been explored for ROS-responsive applications.
Various oxidation-responsive polymersomes have been elabo-
rately constructed from amphiphilic diblock copolymers
bearing phenylboronic ester (PBE) groups in the hydrophobic
block.16–22 The H2O2-mediated degradation of PBE groups
changed the polymersome structure, hence triggering the con-
trolled release of drugs and genes.23,24
main-chain and side-chain polythioethers, such as poly( propy-
lene sulfide),25 poly(L-methionine),26 and S-substituted poly(L-
cysteine).27 Oxidized by H2O2, the transformation of hydro-
phobic thioether to more polar sulfoxide or sulfone signifi-
cantly increases the hydrophilicity of polymers and causes the
disassembly of their assemblies.28 On the other hand,
thioether can complex with metal ions such as iron, mercury,

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silver, and copper,29–32 giving it great potential in constructing
inorganic–polymer hybrid materials. Zhang’s group prepared
copper(I)-coordinated thioether block copolymers with excel-
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lent mechanical and thermal properties and found the melt-
compounded films of the hybrid polymer exhibited a rapid
response to hydrochloric acid vapor and H2O2 solution.33
Stiriba and the co-workers34 utilized the amphiphilic hyper-
branched polyglycerols with thioether shell to complex with
Cu2+, Ag+, and Au3+ ions, and prepared the stable polymer-pro-
tected metal (Cu, Ag, Au) nanoparticles. Long and Chang
reported a thioether-functionalized porous polymer for selec-
tive and efficient Cu2+ capture from biofluids and applied it to
the detection of Wilson’s disease.32
Copper-based nanomaterials have shown promising poten-
tial in ROS-based cancer therapy.35–37 Copper(I) ion has
demonstrated excellent performance for the generation of
strongly oxidative hydroxyl radical (•OH) via a Fenton-like reac-
tion under weakly acidic and neutral conditions, in contrast
with other agents.38,39 Copper(II) ion can deplete overexpressed
GSH inside tumors by a redox reaction. Liu and co-workers
synthesized copper(II)–cysteine mercaptide nanoparticles that
induced ROS generation through the sequential reduction–oxi-
dation reactions with intracellular GSH “AND” H2O2.36 Wang’s
group40 developed a Cu-mediated •OH-generating system
based on a Cu2+–peptide complex with ascorbate, and applied
it in inhibiting cell proliferation and inducing apoptosis
through intra-mitochondrial Fenton reaction. Gai and co-
workers41 constructed copper(II)-based metal–organic frame-
works containing glucose oxidase and demonstrated the
enhanced efficiency of chemodynamic and starvation therapy
via combination with relief hypoxia and GSH depletion. It is
more advantageous to apply Cu2+-based nanomaterials in
cancer treatment because Cu2+ is much more stable than Cu+
in vivo and rapidly reduced to Cu+ by GSH in tumor sites.
In this work, the diblock copolymers containing thioether
and primary amine functionalities were first prepared based
on the reversible addition-fragmentation chain transfer (RAFT)
polymerization of a methionine-based methacrylate monomer
using a poly(ethylene glycol) (PEG)-modified chain transfer
agent. Subsequently, the functionalization of amine groups
generated amphiphilic diblock copolymers PEG-b-P(MET/PBC)
bearing pendant phenylboronic ester carbamate and thioether
moieties in the hydrophobic block. By adjusting the weight
fraction of hydrophobic block in the copolymer, the self-
assemblies formed by PEG-b-P(MET/PBC) underwent a mor-
phological transition from spherical micelles to wormlike
micelles to bilayered polymersomes. In the presence of H2O2,
the generation of polar sulfoxide and free primary amine
groups induced the destruction of the polymersomes into irre-
gular shapes. Taking the metal coordination capability of
thioether into consideration, we investigated the co-assembly
behavior of PEG-b-P(MET/PBC) with the copper(II) ions and
fabricated Cu2+-coordinated hybrid polymersomes. The incor-
poration of Cu2+ ions endows the hybrid polymersomes with
stimuli-responsive behaviors to a competitive ligand and H2O2-
GSH. In the presence of a reducing agent, the hybrid polymer-
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some-mediated Fenton-like reaction gave rise to •OH. The •OH
generation was investigated using terephthalic acid (TPA) as a
detecting probe and by the degradation of methylene blue
(MB), respectively. It is expected that these Cu2+-containing
hybrid polymersomes have potential applications in chemody-
namic cancer therapy.
Experimental section
Materials
Poly(ethylene glycol) monomethyl ether (mPEG45–OH, Mn
2000, 98%) was purchased from Tokyo Chemical Industry
(TCI). 2-Hydroxyethyl methacrylate (HEMA, 98%), N-(tert-butox-
ycarbonyl)-L-methionine (Boc–MET–OH), methylene blue
(MB), terephthalic acid (TPA), and ethylenediaminetetraacetic
acid disodium salt (EDTA) were obtained from Heowns. HEMA
was purified according to a reported procedure.11 Di(N-succini-
midyl) carbonate (98%, Energy Chemical), 4-(hydroxymethyl)
benzeneboronic acid pinacol ester (98%, Energy Chemical),
Triton X-100, dicyclohexylcarbodiimide (DCC, 99%, Sigma-
Aldrich), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, 99%,
Sigma-Aldrich), sodium diethyldithiocarbamate trihydrate
(DDTC, 98%, J&K Scientific), 4-dimethylaminopyridine (DMAP,
99%, J&K Scientific) and 4-cyano-4-(dodecylsulfanylthiocarbo-
nyl)sulfanylpentanoic acid (CDP, 3A Chemicals) were used
directly. 4,4′-Azobis(4-cyanopentanoic acid) (ACPA, 97%,
Sigma-Aldrich) was recrystallized twice from methanol. All sol-
vents were distilled before use. The methionine-based meth-
acrylate monomer Boc–METMA was synthesized by an esterifi-
cation reaction of Boc–MET–OH and HEMA.11 The PEG45-
based chain transfer agent PEG45–CDP was prepared by an
esterification reaction of mPEG45–OH and CDP.11 The phenyl-
boronate containing N-hydroxysuccinimide ester (C1) was pre-
pared according to a reported method (ESI, Fig. S1†).42
Characterizations
1
H NMR spectra were recorded on a Bruker 400 MHz NMR
spectrometer. The number-average molecular weight (Mn),
weight-average molecular weight (Mw), and polydispersity
index (Ð) of the polymers were determined by gel permeation
chromatography (GPC) at 35 °C with a Waters 1525 chromato-
graph equipped with a Waters 2414 refractive index detector.
Tetrahydrofuran (THF) was used as the eluent at a flow rate of
1 mL min−1 and poly(methyl methacrylate) standards were
used for calibration. UV-vis spectroscopy was performed on a
Shimadzu UV-2450 UV-vis spectrophotometer. Fluorescence
spectroscopy was measured on an RF-5301 (Shimadzu) fluoros-
pectrometer. Dynamic light scattering (DLS) analysis was con-
ducted on a Zetasizer Nano ZS from Malvern Instruments
equipped with a 10 mW He–Ne laser at a wavelength of
633 nm at a 90° angle. Zeta potential measurements were con-
ducted on a Zetasizer Nano ZS from Malvern Instruments.
Transmission electron microscopy (TEM) observations were
carried out on a Tecnai G2 20 S-TWIN electron microscope
equipped with a Model 794 CCD camera. The samples were de-
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Polymer Chemistry
posited on a carbon-coated copper grid, and water was evapor-
ated in air. To increase the contrast, the samples were stained
with OsO4 vapor. Scanning electron microscopy (SEM) images
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and elemental mapping analysis were obtained using a ZEISS
MERLIN Compact 616. The concentration of Cu2+ ion was
determined by inductively coupled plasma optical emission
spectrometer (ICP-OES, Spectro-Blue IL).
Synthesis of block copolymers PEG45-b-P(MET/PBC)n
According to our previous work,11 the PEG45-b-P(Boc–
METMA)n diblock copolymers were first prepared by the RAFT
polymerization of Boc–METMA using PEG45–CDP as the
macro-chain transfer agent. The Boc groups in PEG45-b-P(Boc–
METMA)n were removed by treatment with trifluoroacetic acid
(TFA) to obtain the copolymers PEG45-b-P(METMA)n. Next,
phenylboronic ester carbamate (PBC)-functionalized block
copolymers PEG45-b-P(MET/PBC)n were prepared as the follow-
ing. Briefly, PEG45-b-P(METMA)n (150 mg, 0.32 mmol) was dis-
solved in dry THF (3 mL) and then triethylamine (TEA) (67 μL,
0.48 mmol) was added dropwise to the solution in the ice-
water. After the mixture was stirred for 10 minutes, the solu-
tion of the compound C1 (180 mg, 0.48 mmol) in THF (2 mL)
was added. The reaction was carried out at room temperature
for 24 h under Ar atmosphere. The resulted PEG45-b-P(MET/
PBC)n block copolymer was precipitated two times in cold
diethyl ether, centrifuged, and dried under vacuum.
Preparation of PEG45-b-P(MET/PBC)n self-assemblies
Typically, PEG45-b-P(MET/PBC)n (5 mg) was dissolved in 1 mL
of THF, followed by the slow addition of 4 mL of deionized
water. After 2 h stirring, the organic solvent was removed by
dialysis against deionized H2O for 48 h (MWCO 7000). The de-
ionized water was replaced every 4 h.
Preparation of PEG45-b-P(MET/PBC)n/Cu2+ co-assemblies
Briefly, PEG45-b-P(MET/PBC)n (5 mg) was dissolved in 1 mL of
THF, mixed with a predetermined amount of Cu(NO3)2 and
stirred overnight. Then, 4 mL of deionized water was slowly
added to induce the formation of PEG45-b-P(MET/PBC)n/Cu2+ co-
assemblies. After 2 h stirring, the solution was dialyzed against
250 mL of deionized H2O (MWCO 7000) for 48 h without repla-
cing water. An aliquot of solution outside the dialysis bag was
taken out to determine the concentration of free Cu2+ ions by
ICP-OES. The solution of PEG45-b-P(MET/PBC)n/Cu2+ co-assem-
blies was continuously dialyzed against deionized H2O for
another 24 h. The deionized water was replaced every 2 h.
Fluorescence analysis of the exposed primary amine groups
triggered by H2O2
The PEG45-b-P(MET/PBC)40 polymersome solution (0.4 mL, con-
taining 0.76 mg polymer per mL) was incubated with/without
10 mM H2O2 at 37 °C for 24 h. Subsequently, 500 μL of phos-
phate buffer saline (PBS) solution (pH 9.0) and 100 μL of fluo-
roamine (FA) solution in acetone (6 mg mL−1) were added, and
the mixtures reacted for 30 min. The fluorescence emission
spectrum was collected upon excitation at λex = 390 nm.
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Competitive binding with EDTA
The aqueous solution of PEG45-b-P(MET/PBC)26/Cu2+ polymer-
somes (containing 0.38 mg polymer per mL and 0.314 mM
Cu2+ ion) was incubated with different concentrations of EDTA
(0.4 × 10−3, 0.8 × 10−3 and 1.6 × 10−3 M) at room temperature
for 24 h. Then, the mixed solution was dialyzed against water
for 48 h (MWCO 5000). The deionized water was replaced every
4 h.
GSH depletion with the hybrid polymersomes
Ellman’s reagent was used as the indicator to assay the thiol
(–SH) group of GSH. The solution of Ellman’s reagent (0.1 mg
mL−1) was prepared by dissolving DTNB in PBS solution (0.1 M,
pH 8.0). The aqueous solution of GSH (0.2 mM, 0.5 mL) was
incubated with or without PEG45-b-P(MET/PBC)26/Cu2+ polymer-
somes (containing 0.38 mg polymer per mL and 0.314 mM Cu2+
ion) at room temperature for 5 h. Then, the GSH solution was
mixed with 0.5 mL of Ellman’s reagent solution and kept in
dark for 10 min. After adding 1 mL of deionized H2O, the solu-
tion was analyzed by a UV-vis spectrophotometer.
Evaluation of •OH generation
The aqueous solution of PEG45-b-P(MET/PBC)26/Cu2+ polymer-
somes (containing 0.76 mg polymer per mL and 0.628 mM
Cu2+ ion) was mixed with MB solution (3 μg mL−1) and H2O2
solution (10 mM). After the addition of GSH or sodium ascor-
bate (VcNa), the solution was incubated for 12 h at 37 °C. The
change in the absorbance of MB at λ = 660 nm was measured
by a UV-vis spectrophotometer. For the oxidation of TPA, the
PEG45-b-P(MET/PBC)26/Cu2+ polymersomes solution (contain-
ing 0.76 mg polymer per mL and 0.628 mM Cu2+ ion) was
mixed with an equal volume of TPA solution (10 mM) in NaOH
and H2O2 solution (10 mM). After 2 h incubation at 37 °C,
different concentrations of VcNa were added to the solution
and incubated for another 1 h. For GSH, the solution was incu-
bated for another 4 h after the introduction of 2 mM GSH. The
fluorescence emission spectra of the different mixtures were
collected upon excitation at λex = 312 nm.
Results and disscussion
Synthesis and self-assembly of amphiphilic diblock
copolymers PEG45-b-P(MET/PBC)n
The H2O2-responsive phenylboronic ester carbamate (PBC)
functionalities were incorporated into the side chains of the
block copolymer by post-polymerization modification. First,
the diblock copolymers containing pendant thioether and
primary amine groups were prepared via the RAFT polymeriz-
ation of a methionine-derived methacrylate monomer (Boc)–
METMA using PEG45–CDP as the chain transfer agent, fol-
lowed by removal of the Boc groups under an acidic condition
(Scheme 1).11 By adjusting the feeding ratios of (Boc)–METMA
to PEG45–CDP, a series of diblock copolymers with various
P((Boc)–METMA) block lengths, PEG45-b-P((Boc)–METMA)n
(n = 15, 26, 40), were readily prepared by RAFT polymerization,
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Scheme 1 Outline of synthesis of PEG45-b-P(MET/PBC)n block copoly-
mer via RAFT polymerization of methionine-based methacrylate
monomer, and post-polymerization modification through an amidation
reaction.
confirmed by the shift of GPC peak of the block copolymer
toward to a higher molecular weight relative to PEG45–CDP
(Fig. S2†). The average polymerization degree of P((Boc)–
METMA) block was calculated based on the integration ratio of
peak i (CH3O of PEG) and peak b (SCH2 of (Boc)–METMA
units) (Fig. 1a). After the Boc groups were removed by treat-
ment with TFA, the PEG45-b-P(METMA)n block copolymers
with pendant thioether and primary amine groups were
obtained. Successful deprotection was verified by the dis-
appearance of the tert-butyl signal at δ 1.44 ppm and the –NH
signal at δ 5.6 ppm in the 1H NMR spectrum (Fig. 1b).
Subsequently, PBC-functionalized block copolymers were
synthesized through amidation reactions between primary
amine groups of PEG45-b-P(METMA)n and the compound C1
(Scheme 1). The modified block copolymers were characterized
by GPC and 1H NMR. GPC traces revealed the block copoly-
mers after modification had a unimodal molecular weight dis-
tribution and narrow polydispersity (Đ = 1.10–1.23) (Fig. S2†).
Fig. 1 1H NMR spectra of block copolymer PEG45-b-P((Boc)–METMA)n
in CDCl3 (a), PEG45-b-P(METMA)n in D2O (b), and PEG45-b-P(MET/PBC)n
in CDCl3 (c).
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In the 1H NMR spectrum (Fig. 1c), the characteristic signals of
aromatic protons of phenylboronic ester appeared at δ 7.3
( peak r) and 7.8 ppm ( peak s). The signal at 5.1 ppm ( peak n)
was attributed to the methylene adjacent to the benzene ring.
The signal at 1.3 ppm ( peak t) corresponded to the methyl
protons of boronic ester. The signals representing the
thioether groups were observed at 2.6 ppm (SCH2, peak b) and
1.7–2.2 ppm (CH3SCH2CH2, peak a, c). Based on the inte-
gration ratio of the thioether signal at δ 2.6 ppm ( peak b) and
the aromatic proton signal ( peak s) at δ 7.8 ppm, the modifi-
cation degree of METMA units was calculated. The mole frac-
tion of METMA units attached to PBC moieties was calculated
to be about 88% for PEG45-b-P(MET/PBC)15, 91% for PEG45-b-P
(MET/PBC)26, and 93% for PEG45-b-P(MET/PBC)40. The PBC-
functionalized block copolymers are denoted PEG45-b-P(MET/
PBC)n. The structural parameters of PEG45-b-P(MET/PBC)n
block copolymers are listed in Table 1.
The PEG45-b-P(MET/PBC)n block copolymers consist of a
hydrophilic PEG block and a hydrophobic P(MET/PBC) block
containing pendant thioether and phenylboronic ester carba-
mate moieties. These amphiphilic block copolymers are
expected to self-assemble into H2O2-responsive nano-assem-
blies in water. TEM and DLS were utilized to investigate the
effect of hydrophobic block length on the architecture of the
self-assemblies formed by PEG45-b-P(MET/PBC)n block copoly-
mers. The PEG45-b-P(MET/PBC)n self-assemblies were fabri-
cated by adding deionized water to the THF solution of the
polymer, followed by extensive dialysis against water. TEM
images revealed PEG45-b-P(MET/PBC)15 formed spherical
micelles with a diameter of 70–200 nm (Fig. 2a). The spherical
and worm-like micelles coexisted in the TEM image of PEG45-
b-P(MET/PBC)26 assemblies (Fig. 2b). The width of worm-like
micelles was about 29 nm, comparable to the diameter of
spherical micelles. For PEG45-b-P(MET/PBC)40, a well-defined
vesicular structure with a diameter of 230–360 nm was
observed (Fig. 2c). With increasing the length of hydrophobic
P(MET/PBC) block from 15 to 26 to 40, TEM observations indi-
cate PEG45-b-P(MET/PBC)n block copolymers tended to form
nanostructures with decreasing interfacial curvature.43,44 The
resultant self-assemblies undergo a morphological transition,
starting from spherical micelles, progressing to a mixture of
spherical micelles and wormlike micelles, and finally to poly-
mersomes. This morphology evolvement is consistent with the
previous studies on poly(ethylene oxide)-based amphiphilic
diblock copolymers, poly(ethylene oxide)-b-poly(γ-methyl-ε-
caprolactone) and poly(ethylene oxide)-b-poly(N,N-diethyl-
aminoethyl methacrylate).45–47
The DLS results revealed PEG45-b-P(MET/PBC)15 micelles
and PEG45-b-P(MET/PBC)40 polymersomes had a narrow
PDI (0.15–0.16), and the Z-average hydrodynamic
diameters 〈Dh〉 were 248 and 466 nm, respectively (Fig. 2d).
For PEG45-b-P(MET/PBC)26 assemblies, there was a bimodal
size distribution in the DLS curve. The peak at 70–110 nm cor-
responded to spherical micelles, and the other one at
110–1000 nm was attributed to worm-like micelles, in agree-
ment with the TEM observation.
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Table 1 Characterization of PEG45-b-P(MET/PBC)n block copolymers and their self-assemblies
GPC
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Sample Mn (g mol−1)
PEG45-b-P(MET/PBC)15 11 200
PEG45-b-P(MET/PBC)26 14 900
PEG45-b-P(MET/PBC)40 20 500
a
Đ = Mw/Mn. b The Z-average hydrodynamic diameter. c
Observed by TEM (SM = spherical micelles; WM = worm-like micelles; PS =
polymersomes).
Fig. 2 (a–c) TEM images of PEG45-b-P(MET/PBC)n self-assemblies in
the aqueous solution: PEG-b-P(MET/PBC)15 (a), PEG-b-P(MET/PBC)26
(b), and PEG-b-P(MET/PBC)40 (c). (d) DLS curves of PEG45-b-P(MET/
PBC)n self-assemblies in the aqueous solution.
Both thioether and phenylboronic ester carbamate are
H2O2-responsive functionalities. In the presence of H2O2,
thioethers in the P(MET/PBC) block will be converted to polar
sulfoxides, and the oxidation of the boronic ester moieties will
spontaneously initiate a cascade deboronation, 1,6-benzyl
elimination, and decarboxylation reaction to yield p-hydroxy-
benzyl alcohol and liberate the primary amine group
(Scheme 2), thus increasing the hydrophilicity of the PEG-b-P
(MET/PBC) block copolymer. 1H NMR was used to analyze the
variations in the chemical structure of the H2O2-treated copoly-
mer. The block copolymer PEG45-b-P(MET/PBC)15 was selected
Scheme 2 The proposed mechanism for the oxidation responsiveness
of block copolymer PEG45-b-P(MET/PBC)n in the presence of H2O2.
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DLS
Morphologyc
Đa 〈Dh〉b (nm) PDI
1.10 248 0.16 SM
1.17 363 0.27 WM + SM
1.23 466 0.15 PS
for the 1H NMR study due to the good solubility in methanol-d4.
The H2O2 solution (3 μL of 30% aq) was added to the CD3OD-
d4 solution of PEG45-b-P(MET/PBC)15 in an NMR tube. After
4 h reaction at 37 °C, the signals corresponding to aromatic
protons of phenylboronic ester moiety and the signal of
methylene adjacent to the benzene ring almost disappeared in
the 1H NMR spectrum, along with the appearance of the
signals attributed to p-hydroxybenzyl alcohol at δ 7.2, 6.8, and
4.4 ppm (Fig. 3). Moreover, the proton signals representing the
methyl and methylene on sulfoxide were observed at
2.8–3.0 ppm. The 1H NMR result confirms the decomposition
of PBC moieties and transformation of thioethers to sulfox-
ides, triggered by H2O2.
DLS was used to trace the changes in the size of PEG45-b-P
(MET/PBC)n self-assemblies over time at 37 °C in the presence
of 10 mM H2O2 (Fig. S3†). After adding H2O2 to the PEG45-b-P
(MET/PBC)15 micelle solution, DLS data revealed the appear-
ance of small nanoparticles in the range of 20–100 nm after
12 h and much smaller nanoparticles less than 20 nm after
24 h. For PEG45-b-P(MET/PBC)26 worm-like micelles and
PEG45-b-P(MET/PBC)40 polymersomes, the size of the H2O2-
treated assemblies became larger and larger over time. The
DLS results demonstrate the PEG45-b-P(MET/PBC)n self-assem-
blies are H2O2-responsive, and H2O2 can disrupt the structure
of these nano-assemblies.
Next, the oxidation-responsive behavior of PEG45-b-P(MET/
PBC)40 polymersomes was studied by TEM. After 24 h incu-
bation with 10 mM H2O2 at 37 °C, the TEM image revealed
Fig. 3 1H NMR spectra of block copolymer PEG45-b-P(MET/PBC)15
before (a) and after oxidized by H2O2 (b) in methanol-d4.
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Fig. 4 (a and b) TEM images of the PEG45-b-P(MET/PBC)40 polymer-
somes treated with 10 mM H2O2 for 24 (a) and 48 h (b). (c) Fluorescence
spectra of initial PEG45-b-P(MET/PBC)40 polymersomes and oxidized
polymersomes in the presence of fluorescamine (FA) (λex = 390). (d)
Schematic illustration of fluorescence produced by the reaction of FA
with amino-containing molecule.
that the H2O2-treated polymersomes deformed and were in
a swollen state (Fig. 4a). After 48 h, most polymersomes dis-
integrated into an irregular structure (Fig. 4b). In the pres-
ence of H2O2, the generated sulfoxides and the decaged
primary amines in the P(MET/PBC) block induced a hydro-
phobic-to-hydrophilic transition of the membranes of
PEG45 -b-P(MET/PBC)40 polymersomes. Therefore, the
PEG45 -b-P(MET/PBC)40 polymersomes gradually became
swollen over time and eventually disintegrated. The liber-
ation of primary amine groups was confirmed by the amine-
induced emission turn-on of fluorescamine (FA).16 The
mixture of fluorescamine and initial PEG45-b-P(MET/PBC)40
polymersomes was weak fluorescent. However, a strong
emission appeared at λ = 480 nm after FA was introduced
into the H2O2-treated polymersomes (Fig. 4c), indicating
Scheme 3 Illustration of fabrication of hybrid polymersomes with Cu2+-cross-linked membrane through the co-assembly of PEG45-b-P(MET/
PBC)26 and Cu2+, and the stimuli-triggered responsive behavior.
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Polymer Chemistry
the H2O2 -triggered decomposition of PBC moieties released
the primary amine groups.
Fabrication of PEG-b-P(MET/PBC)/Cu2+ hybrid polymersomes
through copper coordination
Taking advantage of the copper–thioether coordination capa-
bility, we investigate the co-assembly of the PEG45-b-P(MET/
PBC)n block copolymers and Cu2+ ions. The formation of
hybrid co-assemblies was induced by adding deionized water
to the mixed solution of the block copolymer and Cu(NO3)2 in
THF, followed by extensive dialysis against water. For PEG45-b-
P(MET/PBC)40, the co-assembly with Cu2+ produced precipi-
tation during the dialysis. The TEM image showed the co-
assembly of PEG45-b-P(MET/PBC)15 with Cu2+ formed spherical
compound micelles with a diameter of about 600 nm
(Fig. S4†). Interestingly, it was found that PEG45-b-P(MET/
PBC)26 co-assembled with Cu2+ to form polymersomes. As
PEG45-b-P(MET/PBC)26 block copolymer self-assembles to a
worm-like structure, the generation of PEG45-b-P(MET/PBC)26/
Cu2+ polymersomes is driven by the coordination interactions
between Cu2+ ions and the block copolymer (Scheme 3).
Besides thioether, the amide nitrogen in MET/PBC unit might
take part in the coordination with Cu2+ ion due to the for-
mation of favorable 6-membered chelate ring.48–50 Keeping the
copolymer concentration constant, various amounts of Cu2+
were added to co-assemble with PEG45-b-P(MET/PBC)26. At
various feeding molar ratios (rf ) of Cu2+ ion and thioether, the
resultant PEG45-b-P(MET/PBC)26/Cu2+ co-assemblies were
denote HP-1 (rf = 0.5), HP-2 (rf = 1), and HP-3 (rf = 2). The free
Cu2+ ions were removed by dialysis against water. After the
amount of free Cu2+ ions was determined by ICP-OES, the
complex ratio of Cu2+ and thioether (rc) was calculated and
listed in Table 2. When the rf varied in the range of 0.5–2.0, rc
increased with the added amount of Cu2+ and approached
0.52 at rf = 2.0.
The morphology of the co-assemblies with various rc values
was observed by TEM. The TEM image of HP-1 co-assemblies
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Table 2 Characterization of PEG45-b-P(MET/PBC)26/Cu2+ assemblies λ 440 nm (Fig. 5d), which is ascribed to the absorption of the
with different copper complex ratios complex formed by DDTC and Cu2+ bound polymer.51 The
elemental mapping analysis from scanning electron
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Sample rf a rc b 〈Dh〉c (nm) Morphologyd ζ (mV)

PEG45-b- 0 — 363 SM + WM −19.1
P(MET/PBC)26
HP-1 0.5 0.23 232 SM + PS 21.5
HP-2 1 0.40 314 SM + PS 17.2
HP-3 2 0.52 415 PS 35.5
a
The feeding ratio of Cu2+ ion and thioether group. b The complex
ratio of Cu2+ ion and thioether group in the co-assemblies.
c
Determined by DLS. d Observed by TEM.
(rc = 0.23) showed the coexistence of spherical micelles
(33–67 nm) and polymersomes (200–360 nm) (Fig. 5a). As rc of
HP-2 co-assemblies increased to 0.4, the number of micelles
decreased, and the polymersomes became larger (Fig. 5b).
With a further increase of rc to 0.52, the Cu2+-containing
hybrid polymersomes (HP-3) became the dormant mor-
phology, and the diameter of most polymersomes was in the
range of 400–500 nm (Fig. 5c). DLS results revealed the 〈Dh〉
gradually increased to 415 nm as rc changed from 0.23 to 0.52
(Fig. S5†), consistent with the TEM observations. These results
indicate that the ratio of Cu2+ ion and thioether has a remark-
able effect on the formation of hybrid polymersomes.
The incorporation of Cu2+ ions switched the surface charge
of the assemblies from a negative value of PEG45-b-P(MET/
PBC)26 micelles (−19.1 mV) to a positive value of hybrid poly-
mersomes. The zeta potential (ζ) value increased with rc and
reached +35.5 mV at rc = 0.52 (Table 2). Moreover, the presence
of Cu2+ ions in polymersomes was demonstrated by the
complex with DDTC reagent. After DDTC was added to the
aqueous solution of the HP-3 polymersomes, the colorless
solution turned yellow-brown ( photo images in Fig. 5d). The
UV-vis spectrum shows an absorption peak centered at
microscopy (SEM) also proved the presence of Cu element on
HP-3 polymersomes (Fig. S6 and S7†). These results confirm
the HP-3 polymersomes are constructed through the complex
of Cu2+ with the PEG45-b-P(MET/PBC)26 block copolymer.
The introduction of metal–ligand interactions endows the
PEG45-b-P(MET/PBC)26/Cu2+ polymersomes with a responsive
property by exchanging the ligand with one that can bind Cu2+
more strongly. EDTA was selected as the chelator in compe-
tition with the block copolymer for complex with Cu2+. The
aqueous solution of EDTA was added to the HP-3 solution
(containing 0.38 mg polymer per mL and 0.314 mM Cu2+ ion).
The final EDTA concentration was 0.4, 0.8, and 1.6 mM,
respectively. After 24 h incubation, the solution was dialyzed
against water. The morphology of EDTA-treated polymersomes
was observed by TEM (Fig. 6a–c). In comparison with the
initial HP-3 polymersomes, the EDTA-treated polymersomes
remained the bilayered vesicular structure but gradually col-
lapsed with increasing EDTA concentrations. After the HP-3
polymersomes were treated with 1.6 mM EDTA, the TEM
image revealed the deformed polymersomes with a decreased
size (150–250 nm). DLS results indicated that the 〈Dh〉 of poly-
mersomes shifted from 415 to 308 nm as the EDTA concen-
tration increased to 1.6 mM (Fig. 6d). The existence of poly-
mersomes indicates that EDTA is not strong enough to com-
pletely destroy the complex between the P(MET/PBC) and Cu2+.
The stability of the polymersomes against Triton X-100 was
investigated by DLS. It is known that the nonionic surfactant
Triton X-100 can transport through the bilayer of polymer-
somes and induce the disassembly of polymersomes.52,53 For
the initial HP-3 polymersomes, the 〈Dh〉 and PDI remained
unaffected after the exposure to Triton X-100 (0.015 M) for
90 min (Fig. 7a). This result also indicated the membrane of

Fig. 5 TEM images of HP-1 (a), HP-2 (b) and HP-3 (c), respectively. Fig. 6 (a–c) TEM images of HP-3 polymersomes treated with various
(d) UV-vis spectra of DDTC, HP-3 polymersomes and DDTC/HP-3 concentrations of EDTA for 24 h: (a) 0.4 mM, (b) 0.8 mM, and (c) 1.6 mM.
polymersomes in water (inset: photo images of the aqueous solutions of (d) DLS curves of HP-3 polymersomes after 24 h treatment with various
DDTC, HP-3 polymersomes, and HP-3/DDTC mixture). concentrations of EDTA.

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Fig. 7 (a) DLS curves of initial HP-3 polymersomes before and after
Fig. 8 (a) Oxidation of TPA in the presence of GSH (HP-3: 0.76 mg
incubated with 0.015 M Triton X-100. (b) DLS curves of EDTA-treated
mL−1, H2O2: 10 mM). (b) Effect of VcNa concentration on the oxidation
HP-3 polymersomes before and after incubated with 0.015 M Triton
of TPA (HP-3: 0.76 mg mL−1, H2O2: 10 mM).
X-100 (EDTA: 1.6 mM).

HP-3 polymersomes was cross-linked by Cu2+–ligand coordi-
nation and hence was impermeable to Triton X-100 molecules.
However, adding Triton X-100 to the 1.6 mM EDTA-treated
polymersomes caused an increase in the 〈Dh〉 (up from 308 to
420 nm) and a broader PDI (Fig. 7b), indicating Triton
X-100 molecules could penetrate the polymersome membrane
and partially solubilize the bilayer. Because of the complex of
EDTA with Cu2+, a fraction of Cu2+ ions leaks out the polymer-
somes, leading to a reduced density of Cu2+-mediated cross-
linking. As a result, the polymersome membrane becomes per-
meable to Triton X-100 to some extent. The DLS result also
demonstrated the incomplete bilayer solubilization by Triton
X-100, which may be linked to the cross-linking caused by the
remained Cu2+ ions inside the polymersome membrane.
Study of Fenton-like reaction mediated by hybrid
polymersomes
Copper(I) ion can efficiently catalyze the Fenton-like reaction
to produce •OH under weakly acidic and neutral
conditions.38,39 However, Cu+ is easily oxidized to Cu2+ in air
because of the low redox potential of Cu2+/Cu+ (∼0.16 V).36 In
this study, we fabricated HP-3 hybrid polymersomes as the
carrier of Cu2+ ions to overcome this problem. Cu2+ ion can be
readily reduced to Cu+ ion by GSH or sodium ascorbate
(VcNa), and subsequently, Cu+ can react with H2O2 to produce
•
OH via a Fenton-like reaction. The reduction of Cu2+ in HP-3
polymersomes was studied by GSH depletion. The Ellman’s
reagent was used as an indicator to assay the thiol group of
GSH. The Ellman’s reagent can react with the thiol of GSH to
yield a product with a characteristic absorbance peak at
λ 412 nm in UV-vis spectrum. The Ellman’s assay revealed the
pure GSH solution exhibited a strong absorbance at λ 412 nm
in the UV-vis spectrum, while the absorbance intensity at
λ 412 nm markedly decreased after GSH solution reacted with
the HP-3 polymersomes for 5 h (Fig. S8†). This result confirms
that the Cu2+ entrapped in the membrane of HP-3 polymer-
somes can be reduced to Cu+ by GSH.
Next, we study the •OH generation performance of HP-3
polymersomes in the aqueous solution containing a reducing
agent and H2O2. Because •OH can oxidize TPA to fluorescent
2-hydroxyterephthalic acid, TPA is used as a probe to detect
the production of •OH by a fluorospectrometer.36,54 For HP-3
polymersomes with 10 mM H2O2 alone, a weak fluorescent peak
of 2-hydroxyterephthalic acid was observed at λ 425 nm (Fig. 8a).
This result indicates the production of few hydroxyl radicals by
the HP-3/H2O2 system due to the following reactions.
Cu2þ þ H2 O2 ! Cuþ þ O2 • ð1Þ
Cuþ þ H2 O2 ! Cu2þ þ OH þ • OH ð2Þ
In contrast, the introduction of 2 mM GSH enhanced the
fluorescent intensity, demonstrating more hydroxyl radicals
were generated through the reduction of Cu2+ ions entrapped
in HP-3 to Cu+ by GSH and subsequent Cu+-catalyzed Fenton-
like reaction (Scheme 3). In the case of sodium ascorbate, an
increase in the fluorescence intensity was observed in a con-
centration range from 0.5 to 3 mM and attenuated when the
concentration reached 5 mM (Fig. 8b). The increase of VcNa
concentration facilitates the generation of more •OH, however,
excess ascorbate ion can quench •OH (k = 1.1 × 1010 M−1 s−1).55
Therefore, the oxidation of TPA is inhibited at the higher con-
centration of VcNa, leading to a decrease in the fluorescence
intensity. The •OH-induced enhancement of fluorescence con-
firmed the •OH generation by the HP-3/reducing agent/H2O2
system through a cascade reduction of entrapped Cu2+ by GSH
(or VcNa) and a Fenton-like reaction.
Next, we investigated the •OH-induced degradation of
methylene blue (MB) under various conditions. As shown in
Fig. 9, MB cannot be degraded by H2O2 alone. In the absence
Fig. 9 Degradation of MB under various conditions (HP-3: 0.76 mg mL−1,
H2O2: 10 mM, VcNa: 1 mM; GSH: 10 mM).

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Polymer Chemistry
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Fig. 10 (a–c) DLS curves of HP-3 polymersomes at specific intervals incubated under various conditions: (a) 1 mM H2O2, (b) 10 mM GSH, and (c)
1 mM H2O2 and 10 mM GSH. (d–f ) TEM images of HP-3 polymersomes after 35 h incubation under various conditions: (d) 1 mM H2O2, (e) 10 mM
GSH, and (f ) 1 mM H2O2 and 10 mM GSH.
of a reducing agent, the absorption of MB at λ 660 nm
decreased to half of the initial absorption after 12 h incubation
with the HP-3/H2O2 system, indicating this system can degrade
a portion of MB molecules. The introduction of a reducing
agent for Cu2+ increased the •OH production, leading to a
remarkable drop in the MB absorption. After 12 h, MB almost
completely degraded in the presence of HP-3, GSH, and H2O2.
DLS and TEM were used to trace the changes in size and
morphology of the HP-3 polymersomes incubated under
various conditions (Fig. 10). In the case of the polymersomes
incubated with 1 mM H2O2 alone, the 〈Dh〉 slightly changed
over time, and the TEM image showed the intact vesicular
structure. In the presence of 10 mM GSH alone, the 〈Dh〉
gradually decreased with time and dropped to 311 nm after
35 h. TEM observation indicated the polymersomes retained
the vesicular nanostructure but became smaller. The reduction
of Cu2+ to Cu+ by GSH leads to the variation of the coordi-
nation structure, resulting in a drop in the polymersome size.
The combination of H2O2 and GSH significantly affected the
size and morphology of the HP-3 polymersomes. DLS curve
showed a broad size distribution and the appearance of small
nanoparticles at 20–110 nm after 25 h. The TEM image dis-
played the HP-3 polymersomes disrupted to a mixture of
spherical micelles and wormlike micelles after 35 h. In the
presence of H2O2 and GSH, the destruction of hybrid polymer-
somes can be ascribed to the cooperation of thioether oxi-
dation and PBC decomposition by H2O2, and Cu2+ reduction
by GSH.
Conclusions
In summary, we synthesized amphiphilic block copolymers
containing pendant phenylboronic ester carbamate and
thioether moieties in the hydrophobic block by RAFT polymer-
ization of a methionine-modified methacrylate monomer, fol-
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lowed by post-polymerization modification. With increasing
the hydrophobic block length, the block copolymer self-
assembled to form nano-assemblies with various mor-
phologies, starting from spherical micelles, evolving to a
mixture of worm-like and spherical micelles, and finally to
polymersomes. The cooperation of thioether oxidation and
PBC decomposition induced the disruption of H2O2-treated
polymersomes to irregular structures. Driven by the coordi-
nation interactions between Cu2+ and the block polymer, the
hybrid polymersomes composed of Cu2+-cross-linked mem-
brane were fabricated via a co-assembly of Cu2+ ions and
the block copolymer. The incorporation of Cu2+ ions
endows the hybrid polymersomes with stimuli-responsive-
ness to EDTA and H2O2−GSH. In the presence of a reducing
agent, the entrapped Cu2+ ions were in situ reduced to Cu+
ions and subsequently initiated the Fenton-like reaction to
produce •OH. The hybrid polymersome-mediated Fenton-like
reaction was evaluated by TPA oxidation and MB degra-
dation. In the presence of H2O2 and GSH, along with the
Fenton-like reaction, the hybrid polymersomes concurrently
disrupted to a mixture of spherical micelles and worm-like
micelles. It is anticipated these Cu2+-containing hybrid
polymersomes have potential applications in the area of
drug delivery.
Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Conflicts of interest
The authors declare no competing financial interest.
Polym. Chem., 2021, 12, 3105–3115 | 3113

Paper
Acknowledgements
This project was financially supported by the National Natural
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Science Foundation of China under contract No. 51773100.
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