Synthesis and self-assembly of thermoresponsive amphiphilic biodegradable polypeptide/poly(ethyl ethylene phosphate) block copolymers

Article abstract of DOI:10.1002/asia.201402524

We report the design and synthesis of new fully biodegradable thermoresponsive amphiphilic poly(γ-benzyl L-glutamate)/poly(ethyl ethylene phosphate) (PBLG-b-PEEP) block copolymers by ring-opening polymerization of N-carboxy-γ-benzyl L-glutamate anhydride (BLG-NCA) with amine-terminated poly(ethyl ethylene phosphate) (H₂N-PEEP) as a macroinitiator. The fluorescence technique demonstrated that the block copolymers could form micelles composed of a hydrophobic core and a hydrophilic shell in aqueous solution. The morphology of the micelles as determined by transmission electron microscopy (TEM) was spherical. The size and critical micelle concentration (CMC) values of the micelles showed a decreasing trend as the PBLG segment increased. However, UV/Vis measurements showed that these block copolymers exhibited a reproducible temperature-responsive behavior with a lower critical solution temperature (LCST) that could be tuned by the block composition and the concentration.

Full text of DOI:10.1002/asia.201402524

FULL PAPER
DOI: 10.1002/asia.201402524
Synthesis and Self-Assembly of Thermoresponsive Amphiphilic
Biodegradable Polypeptide/Poly(ethyl ethylene phosphate) Block
Copolymers
Qiuhua Wu, Dan Zhou, Renyu Kang, Xiuping Tang, Qi Yang, Ximing Song, and
Guolin Zhang*[a]
Abstract: We report the design and
synthesis of new fully biodegradable
thermoresponsive amphiphilic poly(g-
benzyl l-glutamate)/poly(ethyl ethyl-
ene phosphate) (PBLG-b-PEEP) block
copolymers by ring-opening polymeri-
zation of N-carboxy-g-benzyl l-gluta-
demonstrated that the block copoly-
mers could form micelles composed of
a hydrophobic core and a hydrophilic
shell in aqueous solution. The mor-
phology of the micelles as determined
by transmission electron microscopy
(TEM) was spherical. The size and crit-
ical micelle concentration (CMC)
values of the micelles showed a decreas-
ing trend as the PBLG segment in-
creased. However, UV/Vis measure-
ments showed that these block copoly-
mers exhibited a reproducible tempera-
ture-responsive behavior with a lower
critical solution temperature (LCST)
that could be tuned by the block com-
position and the concentration.
À
mate anhydride (BLG NCA) with
Keywords: block copolymers · drug
delivery · peptides · polymers · self-
assembly
amine-terminated poly(ethyl ethylene
À
phosphate) (H2N PEEP) as a macroini-
tiator. The fluorescence technique
Introduction
through changes in temperature.[5] Thermoresponsive poly-
mers are soluble in cold water, although they precipitate
with heating above a certain temperature, known as the
lower critical solution temperature (LCST).[6] This phenom-
enon is reversible; upon cooling the thermosensitive poly-
mers become soluble again.[7] This combination of biode-
gradability made the use of micelles self-assembled from
thermoresponsive polymers possible as new functional bio-
materials to be applied in the construction of novel con-
trolled drug-delivery systems or in other related biomedical
applications.[8]
Polyphosphoesters (PPEs) represent a class of biodegrad-
able polymers with repeated phosphoester attachments in
the backbone, which degrades under the physiological con-
ditions by means of hydrolysis or enzymatic cleavage of the
phosphoester bonds.[9] There is continuous interest in the de-
velopment of PPEs for biomedical applications from drug
and gene delivery to tissue engineering owing to their po-
tential biodegradability, good biocompatibility, and function-
ality of the side chain as well as their structural similarities
to naturally occurring nucleic and teichoic acids.[10] The deg-
radation rates and other physicochemical properties of these
polymers are controlled by the chemical structure in the
backbone and side chains.[11] By choosing biocompatible
building blocks of the polymer, degradation products of
PPEs can have minimal toxic effects and good biocompatibi-
lity.[9c] It has been demonstrated that poly(ethyl ethylene
phosphate) (PEEP), a typical hydrophilic polyphosphoester,
also exhibits good biocompatibility in vitro and in vivo.[12]
Recently, it has been reported that PEEP and its copoly-
mers exhibit thermoresponsibility in aqueous solution.[13]
Micelles fabricated from macromolecular species have at-
tracted considerable interest in contemporary macromolecu-
lar science for both their diversiform morphologies and po-
tential applications.[1] These nanosized assemblies demon-
strate a series of attractive properties in drug-delivery sys-
tems, such as good biocompatibility and high stability in
vitro and in vivo, and have been described as promising ma-
terials in applications such as biosensors, tissue engineering,
or selective drug delivery.[2] Micelles formed by stimuli-re-
sponsive block copolymers in particular have received grow-
ing scientific interest in recent years.[3] Stimuli-responsive
polymers that exhibit unique property changes in response
to environmental stimuli, for example, temperature, pH,
electric fields, and light, are promising for many biomedical
applications, including smart drug/gene-delivery systems, in-
jectable tissue engineering scaffolds, cell culture, and separa-
tion sheets.[4] Among all intelligent polymers studied, tem-
perature-responsive polymeric systems have drawn more at-
tention, because this is an important physiological factor in
the body, and some disease states manifest themselves
[a] Q. Wu, D. Zhou, R. Kang, X. Tang, Q. Yang, X. Song, G. Zhang
Liaoning Province Key Laboratory for Green Synthesis and Prepara-
tive Chemistry of Advanced Materials
College of Chemistry, Liaoning University
No. 66 Chongshan Mid-Road, Huanggu District
Shenyang, Liaoning Province (P.R. China)
Fax : (+86)24-62202380
E-mail: glzhang@lnu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201402524.
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Guolin Zhang et al.
Synthetic polypeptides are known to be very important
Results and Discussion
biodegradable materials. They have the potential to be de-
graded in biological environments. Moreover, since they
have secondary conformations (e.g., a-helices, b-sheets, or
turns), low immunogenicity, good biocompatibility, and ex-
cellent mechanical properties, they may be widely used in
pharmaceuticals and other medical fields. Synthetic polypep-
tides such as poly(g-benzyl-l-glutamate) (PBLG) assumed
a rigid a-helical conformation stabilized by intramolecular
hydrogen-bonding interactions, thereby guaranteeing it to
be a versatile building block and a model for constructing
superstructures and studying the phase behaviors of rod
polymers.[14] But it is well known that most commonly used
polypeptides such as poly(a-leucine), poly(a-alanine),
poly(g-benzyl glutamate), and so on are rather hydrophobic
and degrade very slowly by simple hydrolysis under human
body conditions. To improve the hydrophilicity and to con-
trol the biodegradable rate of these poly(a-amino acid)s,
one might introduce hydrophilic functional groups into the
polymer chain. Poly(ethylene glycol) (PEG) was usually in-
troduced into the copolymer as hydrophilic segment to en-
hance the hydrophilicity of the copolymer because of its ex-
cellent hydrophilicity and biocompatibility.[15] There are
many reports about copolymers of amino acids that were
synthesized from PEG so as to regulate the hydrophilicity
and biodegradable rate, such as poly(aspartic acid)/poly(eth-
ylene glycol) block copolymer, poly(g-benzyl l-glutamate)/
Synthesis of Amine-Terminated Poly(ethyl ethylene
À
À
phosphate) (H2N PEEP)
1
The H NMR spectrum of H2N PEEP is shown in Fig-
ure 1A. The peaks at d=1.36, 3.81, and 4.12 ppm are as-
signed to protons d, c, and e in the PEEP segment, respec-
tively. The peaks at d=3.20 and 4.25 ppm are assigned to
À
protons a and b in the H2NCH2CH2O segment, respective-
ly. The peak at d=3.75 ppm is assigned to proton f in the
methylene proton joined to the end hydroxyl group of the
phosphoester unit of the block copolymer.[6]
poly(ethylene glycol) block copolymer, polyACTHNUGRTENUNG(l-lysine)/poly-
(ethylene glycol) block copolymer, poly(l-alanine)/poly(eth-
ylene glycol) block copolymer, and so on.[16] These amphi-
philic copolymers could be self-assembled into nanoscaled
micelles in a suitable medium, and some have been used as
carriers of drug-delivery systems.[17] However, there are few
reports on fully biodegradable thermoresponsive amphiphil-
ic copolymers that were synthesized from hydrophobic poly-
peptides with hydrophilic and biodegradable products.
Poly(g-benzyl l-glutamate) (PBLG) is one of these synthetic
hydrophobic biodegradable polypeptides, and if the PBLG
chain were combined with PEEP to prepare fully biodegrad-
able thermoresponsive amphiphilic polymers, its hydrophi-
licity and biodegradability should be regulated, and thus its
applications should be extended widely.
In this work, new thermoresponsive amphiphilic biode-
gradable poly(g-benzyl l-glutamate)/poly(ethyl ethylene
phosphate) (PBLG-b-PEEP) block copolymers were synthe-
sized by the ring-opening polymerization of N-carboxy-g-
1
À
Figure 1. H NMR spectra of A) H2N PEEP and B) the PBLG1-b-PEEP
block copolymer in CDCl3.
À
benzyl l-glutamate anhydride (BLG NCA) by using amine-
À
terminated poly(ethyl ethylene phosphate) (H2N PEEP) as
The gel permeation chromatography (GPC) trace of
À
macroinitiator. Their structures were characterized. Then
the PBLG-b-PEEP micelles were prepared by means of the
solvent evaporation method and their physicochemical char-
acteristics and thermosensitivities were investigated.
H2N PEEP is shown in Figure 2A. The sample showed un-
imodal molecular weight distribution, which further indicat-
ed that the polymerization was completed successfully and
there was no another polymer in the product.
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PEEP, thus demonstrating the formation of block polymers.
Molecular weights and molecular weight distributions were
measured by GPC and are summarized in Table 1.
Degradation
The in vitro degradations of PBLG-b-PEEP in phosphate
buffer solution (PBS), pH 7.4, at 378C are shown in the Sup-
porting Information. The introduction of the PEEP segment,
a typical hydrophilic polyphosphoester, into the PBLG
could accelerate the degradation rate. PBLG1-b-PEEP,
PBLG2-b-PEEP, and PBLG3-b-PEEP showed a steady deg-
radation rate, with a 51.1, 39.2, and 28.1% mass loss over
30 days, respectively.
Figure 2. GPC curves of polymers.
Synthesis of Poly(g-benzyl l-glutamate)/Poly(ethyl ethylene
phosphate)/(PBLG-b-PEEP) Block Copolymer
Conformation of Copolymer in Aqueous Solution
It is well known that primary amines, which are more nucle-
ophilic than basic, can be used as initiators for the ring-
opening polymerization of NCA to prepare poly(a-amino
acid)s by undergoing a nucleophilic addition to the carbonyl
The PBLG chain in the copolymers is a synthetic polypep-
tide-like protein. Therefore, it would form a regular confor-
mation such as an a helix or b sheet. The circular dichroism
(CD) spectrum of the PBLG-b-PEEP copolymers in an
aqueous solution is shown in the Supporting Information.
Two negative peaks were clearly observed in the CD curve.
The first one, near 222 nm, was ascribed to an n–p transi-
tion, and the second peak, close to 208 nm, was due to a p–
p transition. Such a pattern of the double negative peaks in
the CD spectrum is typical of an a helix in polypeptides
held together by intramolecular hydrogen-bonding interac-
tions.[19] The result suggested that the PBLG chains
group of NCA.[18] Because H2N PEEP contains a primary
À
amine group, it can initiate ring-opening polymerization of
À
BLG NCA to form block copolymer. A series of block co-
polymers with various molecular weights was synthesized
and the results are summarized in Table 1. It was found that
the total molecular weights of the copolymers increased
along with an increase in the molar ratio of the feeding mo-
À
À
nomer BLG NCA to H2N PEEP.
of the PBLG-b-PEEP copolymers existed in the a-
helix conformation form in the aqueous medium.
Table 1. Related data on PBLG-b-PEEP block copolymers.
[b]
[c]
À
À
Sample
BLG NCA/H2N
WPBLG
/
Mn
Mn
Mw/
Mn
[b]
[c]
PEEP[a]
WPEEP
[gmolÀ1
]
[gmolÀ1
]
À
H2N PEEP
PBLG1-b-
PEEP
–
10/1
–
3040
4790
2886
3989
1.03
1.10
Formation of Micelles
37/63
56/44
67/33
Pyrene has been widely used as a probe to monitor
the association and micellization of macromolecules
in solutions because its photophysical character
changes with variation in the existing environ-
ment.[20] The micellar structures of PBLG-b-PEEP
were confirmed by fluorescence techniques by
using pyrene as a probe. The fluorescence excita-
tion spectra of pyrene in the presence of PBLG1-b-
PBLG2-b-
PEEP
PBLG3-b-
PEEP
20/1
30/1
6980
9260
6125
8167
1.11
1.13
[a] Molar ratio of H2N PEEP to BLG NCA in feed. [b] Determined by 1H NMR
spectroscopy in CDCl3. [c] Determined by GPC in THF at 308C.
À
À
The 1H NMR spectrum of PBLG1-b-PEEP is shown in
Figure 1B. The peaks at d=1.36, 3.81, and 4.12 ppm are as-
signed to protons d, c, and e in the PEEP segment, respec-
tively. The peaks at d=3.20 and 4.25 ppm are assigned to
PEEP at various concentrations are shown in Figure 3A. A
redshift from 334 to 337 nm is observed with increasing con-
centration of PBLG1-b-PEEP, thus indicating that micelliza-
tion takes place for the PBLG-b-PEEP copolymer. Such re-
sults can be attributed to the transfer of pyrene molecules
from water to a hydrophobic environment within the core of
the micelles.
The onset of micellization and the critical micelle concen-
trations (CMCs) could also be obtained from the studies of
excitation spectra.[21] For copolymer PBLG1-b-PEEP, 334
and 337 nm were chosen as the peak wavelength of the
(0,0) band in the pyrene excitation spectra in the aqueous
phase and in the entirely hydrophobic core of the polymeric
micelle, respectively. The pyrene fluorescence intensity
ratios (I337/I334) were plotted against the logarithm of copoly-
À
protons a and b in the H2NCH2CH2O segment, respective-
ly. The peak at d=3.75 ppm is assigned to proton f in the
methylene proton joined to the end hydroxyl group of the
phosphoester unit of the block copolymer. The peaks at d=
2.61, 4.65, 5.04, and 7.29 ppm are assigned to protons h, g, i,
and j in the PBLG segment, respectively. No additional
peaks were detected in the spectrum, thus implying that the
block copolymer was prepared.
The GPC chromatograms of the block copolymers are
shown in Figure 2, and three block copolymers show a unim-
odal peak with decreased retention times relative to H2N
À
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celles are used for systemic drug delivery. Thus, in this
sense, PBLG-b-PEEP micelles should be suitable for phar-
maceutical applications.
To further study the properties of the block copolymer
micelles, both dynamic light scattering (DLS) and TEM
measurements were performed. DLS results showed that all
of the PBLG-b-PEEP micelles exhibited unimodal size dis-
tribution with mean diameters from 87 to 119 nm, and the
detailed data are summarized in Table 2. The sizes of the
micelles decreased with the increase of the proportion of hy-
drophobic segments. The PBLG chain in the copolymer was
a polypeptide-like protein. Therefore, it would form an
a helix by means of intramolecular hydrogen bonding in
aqueous solution (see the Supporting Information). When
the PBLG content increased, the intermolecular hydrogen
bonding was enhanced. When the micellar aggregation
number increased, the density increased and the particle
size decreased.[23] Thus the size of the copolymer micelle
could be adjusted by changing the proportion of the PBLG
segment of the copolymers. The morphologies of the block
copolymer micelles visualized by TEM are shown in Fig-
ure 4B. The copolymer aggregated into approximate spheri-
Figure 3. A) Excitation spectra of pyrene as a function of PBLG1-b-
PEEP concentration in water and B) plots of I337/I334 versus the logarithm
of the PBLG-b-PEEP block copolymer concentrations.
mer concentration. The plots are shown in Figure 3B. Below
a certain concentration, I337/I334 is constant. Above this con-
centration, I337/I334 increased with the increase of logC and
finally reaches a plateau. From this plot, the critical micelle
concentration of 5.75ꢁ10À4 mgmLÀ1 was obtained from the
intersection of two straight lines: the base line and the rap-
idly rising I337/I334 line. The CMCs of PBLG2-b-PEEP and
PBLG3-b-PEEP were also obtained from the same method
and listed in Table 2. The CMC values were in the magni-
Table 2. Related data on PBLG-b-PEEP block copolymer micelles.
[a]
Sample
CMC [mgmLÀ1
]
Diameter [nm][b]
PDI[b]
PBLG1-b-PEEP
PBLG2-b-PEEP
PBLG3-b-PEEP
5.75ꢁ10À4
4.46ꢁ10À4
3.80ꢁ10À4
119
102
87
0.12
0.15
0.14
[a] Critical micelle concentration (CMC) was determined by fluorescence
measurements using pyrene as a probe. [b] Diameter and polydispersity
index (PDI) of micelles were determined by DLS.
tude of 10À4 mgmLÀ1 and reduced with the increase of the
PBLG segment. This is reasonable since the higher content
of the hydrophobic segments will result in stronger interac-
tions between hydrophobic chains, thus leading to a more
stable structure and therefore to a lower CMC value. This
trend is in agreement with the results reported in the litera-
ture.[6,22] The low CMC values of PBLG-b-PEEP micelles
might be due to the hydrophobicity and the a-helix confor-
mation form of the PBLG block in aqueous medium. There-
fore, it is believed that PBLG-b-PEEP micelles would be
thermodynamically stable in aqueous media, and lower
CMC values are surely a favorable indication when such mi-
Figure 4. Characterization of the PBLG-b-PEEP micelles. A) The size
distribution of PBLG-b-PEEP micelles determined by DLS:
A1) PBLG1-b-PEEP, A2) PBLG2-b-PEEP, and A3) PBLG3-b-PEEP mi-
celles. B) TEM images of self-assembled micelles: B1) PBLG1-b-PEEP,
B2) PBLG2-b-PEEP, and B3) PBLG3-b-PEEP micelles (c=0.1 mgmLÀ1
258C).
,
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cal micelles in aqueous solution, and the micelle sizes as de-
termined by TEM were smaller than those determined by
DLS in Figure 4A, because the micelle diameters deter-
mined by DLS represent their hydrodynamic diameter,
whereas those obtained by TEM are related to the collapsed
micelles after water evaporation.
utions of PBLG1-b-PEEP micelles at different concentra-
tions. We can clearly see that LCST values increase as the
block copolymer concentration decreases, and that the
lower the polymer concentration is, the broader the temper-
ature range exhibited by the decrease in transmittance. Fig-
ure 5B illustrates the effects of the block copolymer concen-
trations on LCST values for PBLG1-b-PEEP samples. As
the polymer concentrations increase from 0.05 to
0.5 mgmLÀ1, LCST values decrease from 37 to 298C for
PBLG1-b-PEEP. In addition, it should be noted that the
sharpness of the thermally induced phase transition was de-
pendent on the polymer concentration. This finding is con-
sistent with the generally accepted LCST principle for dilute
solutions, which states that higher water content enhances
the hydrogen-bonding interactions between water and the
polymer chain, and this requires more thermal energy to
break the water structure, thereby resulting in an increase in
the LCST.[25]
It is reported that the LCST of thermoresponsive poly-
mers can be controlled by compositions of hydrophobic and
hydrophilic units.[26] In this study, the LCST transition be-
haviors of the PBLG-b-PEEP block copolymers were tuned
by changing the lengths of the hydrophobic PBLG block:
the larger the hydrophobic block content of the copolymer,
the lower the LCST at a fixed PEEP molecular weight. Fig-
ure 6A shows the temperature dependence of optical trans-
Thermosensitivity of Polymers
The thermal phase transition temperature, expressed as the
lower critical solution temperature (LCST), is one of the
basic physical properties of thermoresponsive water-soluble
polymers. The Supporting Information shows a typical pho-
tograph of aqueous solutions of PBLG1-b-PEEP. Below the
LCST, PBLG-b-PEEP copolymers are amphiphilic and con-
sist of a hydrophobic block (PBLG) and hydrophilic block
(PEEP). The solution was transparent and colorless (see the
Supporting Information). However, when heated close to
the LCST, the solution gradually turned into a semitranspar-
ent emulsion (see the Supporting Information); above the
LCST, the copolymers were hydrophobic and the solution
became a white opaque suspension (see the Supporting In-
formation); and finally the polymers precipitated from
water if the solution was kept at a high temperature for
enough time. When cooled, the semitransparent emulsion
and transparent colorless solution were obtained again. Evi-
dently, PBLG1-b-PEEP showed a reversible LCST phase
transition in water. This phenomenon takes place on ac-
count of the different solvation of PEEP chains by water
molecules at the temperatures below and above the phase-
transition temperature.[24]
Figure 5A shows the temperature-dependent transmit-
tance at a wavelength of 500 nm obtained from aqueous sol-
Figure 6. A) Effect of hydrophobic block length on the thermoresponsive
behavior of copolymers in aqueous solution (c=0.1 mgmLÀ1
) and
B) LCST as a function of the PBLG block in the copolymers.
mittance of micellar solutions of the copolymers with differ-
ent hydrophobic PBLG block lengths. The transmittance de-
creased significantly at a specific temperature upon heating
solutions of all of the copolymers. Figure 6B illustrates the
effects of the PBLG wt% on LCST values for PBLG-b-
PEEP copolymers. As the PBLG wt% increased from 0 to
Figure 5. A) Temperature dependences of optical transmittance at
500 nm obtained for aqueous solutions of PBLG1-b-PEEP at varying
polymer concentrations and B) LCST as a function of the concentration
of PBLG1-b-PEEP.
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67%, LCST values decreased from 39 to 288C, thus showing
a decreasing trend as the PBLG block length increased. The
LCSTs of PBLG-b-PEEP polymers were found to be lower
when the temperature increased from 22 to 458C and re-
verted to the transparent state when the temperature of the
solution decreased from 45 to 228C. The highest and lowest
transmittance of the PBLG1-b-PEEP micellar solution re-
mained almost constant during multiple heating/cooling
cycles without any detectable hysteresis. However, a hystere-
sis was observed for copolymers PBLG2-b-PEEP and
PBLG3-b-PEEP with a greater proportion of hydrophobic
PBLG, as shown in Figure 7B. The hysteresis was due to the
fact that greater hydrophobic character of the copolymer
leads to the formation of more stable and larger aggregates
by means of intermicelle association, in which the larger hy-
drophobic domain and closely compact structure could pre-
vent PEEP rehydration by water upon cooling. Thus, the
larger aggregates dissociated slowly to individual micelles.[28]
The phase-transition behaviors of thermoresponsive poly-
mers in aqueous solution are known to be influenced by ad-
ditives such as salts, surfactants, and alcohols. The reason is
that these additives can alter the interactions between the
polymer and water.[29] As a typical water structure-maker,
sodium chloride can disrupt the hydration structure sur-
rounding the polymer chains, thus leading to a salting-out
effect and thereby lowering the LCST of the polymer solu-
tion. Figure 8 shows the influence of the NaCl concentration
À
than H2N PEEP homopolymers and to depend on their mo-
lecular weights. This was considered to result from hydrating
contributions from polar terminal hydrophilic amino groups
in the copolymers. In general, the LCST decreased as the
hydrophilicity of the polymer decreased. Considering the
fact that the PBLG block is more hydrophobic than the
PEEP block, it is reasonable that the LCST decreases as the
molar fraction of PBLG increases. These results clearly
show that the phase transition of PBLG-b-PEEP can be
controlled within a temperature range (28–398C) by varying
the block length of the PBLG block. Similar observations
have been reported for several thermosensitive polymers.[27]
Thermoresponsivity under physiological conditions is effec-
tive for drug delivery or tissue engineering applications. In
fact, the LCST of thermoresponsive polymers can be con-
trolled by compositions of hydrophobic and hydrophilic
units.[13] Thus, LCST can also be further tuned by adjusting
the composition of the PBLG block or by adding new hy-
drophobic blocks to find its application under physiological
temperatures.
The phase-transition behavior of PBLG1-b-PEEP during
the heating/cooling cycle was monitored by measuring the
change of the transmittance with an increment of 1.08C, as
shown in Figure 7A. An interesting observation is the excel-
lent reversibility and reproducibility displayed by the copo-
lymer PBLG1-b-PEEP in the optical transmittance of the
micellar solution during 10 cycles of temperature change be-
tween 228C (below the LCST) and 458C (above the LCST).
The transparent micellar solution rapidly became cloudy
Figure 8. Effect of NaCl concentration on the LCST of a micellar solution
of PBLG1-b-PEEP (c=0.1 mgmLÀ1).
on the thermosensitivity of PBLG1-b-PEEP by measuring
the transmittance of polymer micelles in aqueous solution.
With the increase in NaCl concentration, a decrease in
LCST was observed, thus indicating a typical salting-out
effect. It is explained by the fact that the presence of NaCl
increases the hydrogen bonding among water molecules and
decreases the hydrogen bonding among water and hydro-
philic chains, which might lead to a partial dehydration of
PEEP and results in a stronger tendency of association of
PEEP that decreases its LCST. However, the addition of
NaCl will undoubtedly increase the polarity of aqueous
media and thus enhance the hydrophobic–hydrophobic in-
teraction and subsequently lead to the stronger tendency for
copolymer association, which is indicated by the decrease in
LCST. This trend was in agreement with the result reported
in the literature.[9e,28a]
Figure 7. A) Reversible changes of optical transmittance in response to
temperature fluctuations for PBLG1-b-PEEP micellar solution (c=
0.1 mgmLÀ1) and B) transmittance versus temperature plots for various
copolymer aqueous solutions (c=0.1 mgmLÀ1) during one heating–cool-
ing cycle.
Figure 9 shows the changes in the hydrodynamic diameter
(dH) for PBLG1-b-PEEP aqueous solution during the heat-
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Conclusion
In the present study, new thermosensitive amphiphilic bio-
degradable PBLG-b-PEEP block copolymers with various
PBLG and PEEP block lengths were synthesized. The struc-
tures and compositions of the polymers were characterized
1
by H NMR spectroscopy and GPC. These copolymers had
low CMC values that ranged from 3.80 to 5.75ꢁ
10À4 mgmLÀ1. The behaviors of the block copolymer in
aqueous solution were also studied by DLS and TEM.
These block copolymers were able to spontaneously self-as-
semble into micelles of around 87–119 nm in size in aqueous
solution, which contained a hydrophobic PBLG core and
hydrophilic PEEP shell. It has been revealed that the phase
transition of PBLG-b-PEEP micelles is reversible, and the
thermosensitivity is affected by the molecular weight, com-
position of the PBLG block, and the sodium chloride con-
centration in the medium, which in turn allows convenient
adjustment of their thermosensitivity.
Figure 9. The changes in the hydrodynamic diameter (dH) for PBLG1-b-
PEEP aqueous solution during the heating process (c=0.1 mgmLÀ1).
ing process. Three clear regions can be observed: a) first the
equilibrium stage, then b) the sharp increase, and c) the
final decrease. At low temperature, the size of the micelle
stays the same, almost without a clear response to the tem-
perature. As the solution is heated to approximately 348C,
a clear phase transition takes place, and dH increases rapidly
to 715 nm owing to the aggregation of the micelles, thereby
revealing that the thermoresponsive PEEP shell probably
leads to high assembly by means of the intra- and intermi-
celle hydrogen bonds. However, above the transition tem-
perature, with the temperature increasing persistently, the
dH value goes through a decrease, which might result from
the collapse of the aggregates by the removal of more water
molecules and the formation of more compact and regular
structures.[30]
Experimental Section
Materials
l-Glutamic acid (biochemical grade) was purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China) and dried under vacuum
for 24 h before use. Triphosgene (chemical reagent) was obtained from
Haining Zhonglian Chemical Reagent Co., Ltd. (Zhejiang, China) and
was used without any treatment. Stannous octoate (SnACTHUNRGTNEUNG(Oct)2) was pur-
chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
tert-Butoxycarbonyl (Boc)-aminoethanol (Boc-NHCH2CH2OH; analyti-
cal reagent) was purchased from Tokyo Kasei Kogyo Co., Ltd. (TCI).
Tetrahydrofuran (THF; analytical grade) was purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China), dried, and distilled in the
presence of sodium immediately before use. Phosphorus trichloride (ana-
lytical grade) was purchased from Guangfu Fine Chemical Research In-
stitute (Tianjin, China) and distilled before use. Ethylene glycol (analyti-
cal grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China), dried over MgSO4, and distilled under vacuum before
use. Toluene (analytical grade) was purchased from Baishi Chemical Re-
agent Co., Ltd. (Tianjin, China), dried, and distilled in the presence of
sodium immediately before use. Triethylamine (TEA; analytical grade)
was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China), dried over CaH2, and distilled before use. Dichloromethane
(CH2Cl2, analytical grade) was supplied by Sinopharm Chemical Reagent
Co., Ltd. (Shanghai, China), dried over P2O5, and distilled before use.
Other chemicals are all analytical reagents made in China and used with-
out further purification.
Scheme 1 shows a schematic illustration of thermally in-
duced aggregation and phase transition of PEEP-b-PBLG
Scheme 1. Schematic illustration of the thermally induced aggregation
and phase transition of PBLG-b-PEEP copolymers in water.
copolymers in water. When the solution temperature was
below the LCST (stage 1), spherical polymer micelles exist-
ed individually and the micellar solution was clear (see the
Supporting Information). At temperatures close to the
LCST (stage 2), intermicelle aggregation gave rise to the
formation of larger aggregates with a multicore structure
and associated with an abrupt increase in the aggregate
radius. Moreover, the solution became cloudy (see the Sup-
porting Information). When the temperature continued to
increase, the collapse of the aggregates occurred by the re-
moval of more water molecules and the formation of more
compact and regular structures (Stage 3). The micelle dehy-
dration certainly increased the solution turbidity on account
of the scattering (see the Supporting Information).
Synthesis of Ethyl Ethylene Phosphate (EEP)
Ethyl ethylene phosphate (EEP) was prepared according to the litera-
ture.[31] Briefly, ethylene glycol (124.14 g, 2 mol) was added dropwise to
a stirred mixture of phosphorus trichloride (274.66 g, 2 mol) and dry
CH2Cl2 (250 mL). After complete addition of ethylene glycol, the solu-
tion was stirred at room temperature for another 0.5 h, and CH2Cl2 was
evaporated under reduced pressure. The residue was distilled under re-
duced pressure to give 2-chloro-1,3,2-dioxaphospholane (123.2 g). Yield:
49%, b.p. 42–458C/1600 Pa.
The oxidation of 2-chloro-1,3,2-dioxaphospholane (123.2 g) was carried
out by bubbling O2 through the solution in toluene at 408C for 48 h.
After removal of benzene, the residue was distilled under reduced pres-
sure to give 2-chloro-2-oxo-1,3,2-dioxaphospholane (77.9 g) as a colorless
liquid. Yield: 56%, b.p. 88–908C/107 Pa.
Chem. Asian J. 2014, 9, 2850 – 2858
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Guolin Zhang et al.
A mixture of anhydrous ethanol (25.3 g, 0.55 mol) and anhydrous trie-
thylamine (61.6 g, 0.605 mol) was added dropwise to a stirred and cooled
mixture (À58C) that contained 2-chloro-2-oxo-1,3,2-dioxaphospholane
(77.9 g, 0.55 mol) and anhydrous toluene (250 mL). The temperature of
the reaction mixture was maintained at À58C. After complete addition,
the resulting mixture was stirred at room temperature for another 2 h.
Thereafter, the triethylamine hydrochloride was filtered off and the fil-
trate was concentrated. The residue was distilled under reduced pressure
to give ethyl ethylene phosphate (EEP; 90.5 g) as a colorless liquid.
Yield: 61%, b.p. 95–978C/107 Pa.
Synthesis of Poly(g-benzyl l-glutamate)/Poly(ethyl ethylene phosphate)/
(PBLG-b-PEEP) Block Copolymer
À
À
Certain amounts of H2N PEEP and BLG NCA were dissolved together
in anhydrous CH2Cl2 (40 mL) and kept at 258C for 24 h under stirring.
The reaction mixture was poured into ethyl ether (250 mL) to give
a white precipitate. The precipitate was dried under vacuum at 308C for
12 h to give the desired PBLG-b-PEEP block copolymers. The yield was
approximately 50%. The reaction route is shown in Scheme 2. Different
À
À
molar ratios of the feeding BLG NCA to H2N PEEP resulted in the
corresponding copolymers with various compositions as listed in Table 1.
À
Synthesis of N-carboxy-g-benzyl l-glutamate Anhydride (BLG NCA)
In Vitro Degradations of Copolymers
g-Benzyl l-glutamate was prepared from l-glutamic acid and benzyl alco-
hol (Scheme 1). M.p. 172–1748C;[32] yield: 44.1%; elemental analysis
calcd (%) for C12H15NO4: C 60.75, H 6.37, N 5.90; found: C 60.48, H
The in vitro degradation study was conducted by placing the disk samples
described above in PBS (5 mL, 0.1 molLÀ1), pH 7.4 at 378C. The samples
were with drawn at predetermined time points, washed three times with
ultrapurified water, and dried to constant weight under vacuum.
À
6.62, N 5.97. N-Carboxy-g-benzyl l-glutamate anhydride (BLG NCA)
was prepared by the reaction of g-benzyl l-glutamate with triphosgene in
dried THF at 508C according to a literature procedure. M.p. 96–978C;[33]
yield: 42.2%; elemental analysis calcd (%) for C13H13NO5: C 59.31, H
4.98, N 5.32; found: C 59.54, H 4.87, N 5.26. 1H NMR (CDCl3): d=2.08,
Preparation of Micelles
The micelles were prepared using a solvent displacement method with
a tetrahydrofuran/water (THF/H2O) system.[34] A copolymer (50 mg) was
first dissolved in THF (10 mL); thereafter the copolymer solution was
slowly added into ultrapurified water (30 mL; Aquaplus 18.2 MW). THF
was removed using a rotary evaporator at 258C for 2 h. The obtained so-
lution was transferred into a 50 mL volumetric flask, followed by dilution
to the calibration mark with ultrapurified water to obtain 1 mgmLÀ1 mi-
celles.
À
À
2.29 (2H; CH2OOCCH2CH2), 2.57 (2H; CH2OOCCH2CH2), 4.38 (1H;
À
À
CH), 5.13 (2H; CH2OOCCH2CH2), 6.52 (1H; NH), 7.37 ppm (5H;
À
Ar H). The reaction route is shown in Scheme 2.
À
Synthesis of H2N PEEP
EEP (3.003 g, 19.8 mmol) and THF (10 mL) were transferred to a 50 mL
round-bottomed flask. After six cycles of evacuation purging with puri-
fied nitrogen, SnACHTUNGTRENNUNG(Oct)2 (0.201 g, 0.49 mmol) and Boc-aminoethanol
Characterization
(0.074 g, 0.45 mmol) were added to the round-bottomed flask and kept at
408C for 3 h under stirring. After THF was removed under reduced pres-
sure, the resulting polymer was dissolved in anhydrous CH2Cl2 (5 mL),
precipitated in petroleum ether (100 mL), and kept at 28C for 12 h. The
precipitate was dried under vacuum at 308C for 48 h to give the desired
Elemental analysis was performed with a Thermo Electron Flash EA
1112 instrument. H NMR spectra were measured with a Varian Mercury
1
300 NMR spectrometer at room temperature using CDCl3 as solvent.
Chemical shifts (d) are given in ppm using tetramethylsilane (TMS) as an
internal reference. GPC measurements were conducted with a Waters
1515 GPC instrument equipped with an HT4 and HT3 column (effective
molecular-weight range: 5000 to 600000 and 500 to 30000) and a 2414
differential refractive index detector. THF was used as eluent at the flow
rate of 1.0 mLminÀ1 at 308C, and the molecular weights were calibrated
with polystyrene standards. The circular dichroism (CD) spectrum in an
aqueous solution was measured with a JASCO 810 spectrophotometer.
The CMCs of the copolymers were determined by fluorescence measure-
ments using pyrene as a probe. A pyrene solution (in acetone) was added
into a series of volumetric flasks in such an amount that the final concen-
tration of pyrene in each solution was 6.5ꢁ10À7 molLÀ1; thereafter ace-
tone was removed completely. The
À
BocNH PEEP. Yield: 40%.
À
H2N PEEP was prepared by the removal of the Boc group from
À
À
BocNH PEEP. Typically, BocNH PEEP was dissolved in CH2Cl2
(10 mL) and treated with trifluoroacetic acid (2 mL) at 08C for 1 h under
stirring. Then the solution was poured into n-hexane (100 mL) and kept
at 28C for 12 h. The precipitate was dried in vacuum at 308C for 48 h to
À
give the desired H2N PEEP. Yield: 80%. The reaction route is shown in
Scheme 2.
polymer solution was added into the
volumetric flasks and diluted till the
calibration mark using ultrapurified
water to obtain the desired copolymer
concentrations, which ranged from
1.0ꢁ10À6 to 1.0 mgmLÀ1. The samples
were maintained at 508C for 2 h and
stored at room temperature overnight
to equilibrate pyrene and micelles.
Steady-state fluorescence excitation
spectra were recorded with a Varian
Cary Eclipse fluorescence spectropho-
tometer at 390 nm emission wave-
length and 2.5 nm slit width. The scan
rate was 120 nmminÀ1. The size distri-
bution of micelles was determined by
dynamic light scattering with a Malvern
Nano ZS instrument at 258C. The
morphology of the micelles was inves-
tigated by TEM, which was carried
out with a Hitachi H-7650 electron mi-
croscope operating at an accelerating
voltage of 80 kV. Specimens were pre-
Scheme 2. The synthesis of the PBLG-b-PEEP block copolymer.
Chem. Asian J. 2014, 9, 2850 – 2858
pared by transferring a drop of the mi-
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Guolin Zhang et al.
celle solution onto a 200 mesh copper grid coated with carbon and allow-
ing the sample to dry in air before measurements. The lower critical solu-
tion temperature of the aqueous solution of the polymer was investigated
with a Perkin–Elmer Lambda Bio 20 UV/Vis spectrophotometer together
with a NESLAB RTE-111 temperature controller. Briefly, the polymers
were dispersed in ultrapurified water (Aquaplus 18.2 MW). The transmit-
tance of aqueous solutions of polymer at l=500 nm was recorded in
a 1.0 cm path length quartz cell. In the heating/cooling cycle, the rate of
heating or cooling was set at 18CminÀ1 with hold steps of 10 min at each
temperature. Values for the LCST of aqueous solutions of the polymers
were determined at a temperature with a half of the optical transmittance
between the below and above transitions.
2006, 27, 1027–1034; d) Z. Zhao, J. Wang, H. Q. Mao, K. W. Leong,
Adv. Drug Delivery Rev. 2003, 55, 483–499.
[11] L. S. Naira, C. T. Laurencina, Prog. Polym. Sci. 2007, 32, 762–798.
[12] a) Y. Iwasaki, E. Yamaguchi, Macromolecules 2010, 43, 2664–2666;
b) J. Cheng, J. X. Ding, Y. C. Wang, J. Wang, Polymer 2008, 49,
4784–4790; c) Y. Y. Yuan, Q. Du, Y. C. Wang, J. Wang, Macromole-
cules 2010, 43, 1739–1746.
[13] Y. Iwasaki, C. Wachiralarpphaithoon, K. Akiyoshi, Macromolecules
2007, 40, 8136–8138.
ˇ
[14] a) F. Rypꢂcek, Polym. Degrad. Stab. 1998, 59, 345–351; b) H. Iwata,
S. Matsuda, K. Mitsuhashi, E. Itoh, Y. Ikada, Biomaterials 1998, 19,
1869–1876; c) T. Fukuoka, H. Uyama, S. Kobayashi, Biomacromole-
cules 2004, 5, 977–983; d) A. Wamsley, B. Jasti, P. Phiasivongsa,
X. L. Li, J. Polym. Sci. Part A 2004, 42, 317–325; e) C. Y. Chen,
Z. H. Wang, Z. B. Li, Biomacromolecules 2011, 12, 2859–2863; f) Y.
You, Y. Chen, C. Hua, C. M. Dong, J. Polym. Sci. Part A 2010, 48,
709–718.
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (grant no. 51373073), the Natural Science Founda-
tion for Education Department of Liaoning Province of China (grant no.
L2012007), the Program for Liaoning Innovative Research Team in Uni-
versity (grant no. LT2011001), and the Foundation of 211 Project for In-
novative Talents Training, Liaoning University.
[15] A. Lavasanifar, J. Samuel, G. S. Kwon, Adv. Drug Delivery Rev.
2002, 54, 169–190.
[16] a) A. Harada, K. Kataoka, Macromolecules 1998, 31, 288–294; b) K.
Kugo, A. Ohji, T. Uno, J. Nishino, Polym. J. 1987, 19, 375–381; c) A.
Harada, K. Kataoka, Macromolecules 1995, 28, 5294–5299; d) G. L.
Zhang, J. B. Ma, Y. H. Li, Y. N. Wang, J. Biomater. Sci. Polym. Ed.
2003, 14, 1389–1400.
[17] a) Y. Li, G. S. Kwon, Pharm. Res. 2000, 17, 607–611; b) K. Kataoka,
T. Matsumoto, M. Yokoyama, T. Okamo, Y. Sakurai, S. Fukushima,
K. Okamoto, G. S. Kwon, J. Controlled Release 2000, 64, 143–153.
[18] Y. Yamashita, Y. Iwaya, K. Ito, Makromol. Chem. 1975, 176, 1207–
1216.
[1] a) H. Kuramochi, Y. Andoh, N. Yoshii, S. Okazaki, J. Phys. Chem. B
2009, 113, 15181–15188; b) J. X. Zhang, K. Ellsworth, P. X. Ma, J.
Controlled Release 2010, 145, 116–123; c) C. H. Cai, J. P. Lin, T.
Chen, X. H. Tian, Langmuir 2010, 26, 2791–2797.
[2] a) C. L. Peng, M. J. Shieh, M. H. Tsai, C. C. Chang, P. S. Lai, Bioma-
terials 2008, 29, 3599–3608; b) X. W. Zhang, M. Oddon, O. Giani, S.
Monge, J. J. Robin, Macromolecules 2010, 43, 2654–2656; c) Y. Hu,
Z. P. Jiang, R. Chen, W. Wu, X. Q. Jiang, Biomacromolecules 2010,
11, 481–488.
[19] a) G. Holzwarth, P. Doty, J. Am. Chem. Soc. 1965, 87, 218–228;
b) D. C. Dong, M. A. Winnik, Can. J. Chem. 1984, 62, 2560–2565.
[20] T. Li, J. P. Lin, T. Chen, S. N. Zhang, Polymer 2006, 47, 4485–4489.
[21] C. Deng, X. S. Chen, H. J. Yu, J. Sun, T. C. Lu, X. B. Jing, Polymer
2007, 48, 139–149.
[3] a) J. Yao, Y. L. Ruan, T. Zhai, J. Guan, G. P. Tang, H. R. Li, S. Dai,
Polymer 2011, 52, 3396–3404; b) Y. Y. Li, X. Z. Zhang, H. Cheng,
J. L. Zhu, S. X. Cheng, R. X. Zhuo, Macromol. Rapid Commun.
2006, 27, 1913–1919; c) J. Virtanen, H. Tenhu, J. Polym. Sci. Part A
2001, 39, 3716–3725.
[4] a) Y. C. Wang, L. Y. Tang, Y. Li, J. Wang, Biomacromolecules 2009,
10, 66–73; b) J. Bigot, B. Charleux, G. Cooke, F. Delattre, D. Four-
nier, J. Lyskawa, L. Sambe, F. Stoffelbach, P. Woisel, J. Am. Chem.
Soc. 2010, 132, 10796–10801; c) A. Kumar, A. Srivastava, I. Y.
Galaev, B. Mattiasson, Prog. Polym. Sci. 2007, 32, 1205–1237.
[5] a) T. H. Qu, A. R. Wang, J. F. Yuan, J. H. Shi, Q. Y. Gao, Colloids
Surf. B 2009, 72, 94–100; b) S. T. Sun, P. Y. Wu, J. Phys. Chem. B
2011, 115, 11609–11618.
[6] X. Liu, P. H. Ni, J. L. He, M. Z. Zhang, Macromolecules 2010, 43,
4771–4781.
[7] H. Vihola, A. Laukkanen, H. Tenhu, J. Hirvonen, J. Pharm. Sci.
2008, 97, 4783–4793.
[8] S. Ganta, H. Devalapally, A. Shahiwala, M. Amiji, J. Controlled Re-
lease 2008, 126, 187–204.
[9] a) Y. C. Wang, Y. Y. Yuan, F. Wang, J. Wang, J. Polym. Sci. Part A
2011, 49, 487–494; b) N. Koseva, A. Bogomilova, K. Atkova, K.
Troev, React. Funct. Polym. 2008, 68, 954–966; c) D. P. Chen, J.
Wang, Macromolecules 2006, 39, 473–475; d) C. S. Xiao, Y. C.
Wang, J. Z. Du, X. S. Chen, J. Wang, Macromolecules 2006, 39,
6825–6831; e) Y. C. Wang, Y. Li, X. Z. Yang, Y. Y. Yuan, L. F. Yan,
J. Wang, Macromolecules 2009, 42, 3026–3032; f) J. L. He, P. H. Ni,
S. Wang, H. Y. Shao, M. Z. Zhang, X. L. Zhu, J. Polym. Sci. Part A
2010, 48, 1919–1930.
[10] a) J. Wang, H. Q. Mao, K. W. Leong, J. Am. Chem. Soc. 2001, 123,
9480–9481; b) C. Wachiralarpphaithoon, Y. Iwasakia, K. Akiyoshi,
Biomaterials 2007, 28, 984–993; c) Q. Li, J. Wang, S. Shahani,
D. D. N. Sun, B. Sharma, J. H. Elisseeff, K. W. Leong, Biomaterials
[22] Y. C. Wang, L. Y. Tang, T. M. Sun, C. H. Li, M. H. Xiong, J. Wang,
Biomacromolecules 2008, 9, 388–395.
[23] G. L. Zhang, F. Liang, X. M. Song, D. L. Liu, M. J. Li, Q. H. Wu,
Carbohydr. Polym. 2010, 80, 885–890.
[24] J. Hain, M. Schrinner, Y. Lu, A. Pich, Small 2008, 4, 2016–2024.
[25] a) L. D. Shao, M. Q. Hu, L. Chen, L. Xu, Y. M. Bi, React. Funct.
Polym. 2012, 72, 407–413; b) S. Verbrugghe, K. Bernaerts, F. E.
Du Prez, Macromol. Chem. Phys. 2003, 204, 1217–1225.
[26] S. Shah, A. Pal, R. Gude, S. Devi, Eur. Polym. J. 2010, 46, 958–967.
[27] a) H. Vihola1, A. Laukkanen, L. Valtola, H. Tenhu, J. Hirvonen,
Biomaterials 2005, 26, 3055–3064; b) Y. Maeda, M. Tsubota, I.
Ikeda, Macromol. Rapid Commun. 2003, 24, 242–245.
[28] a) Q. L. Cui, F. P. Wu, E. J. Wang, J. Phys. Chem. B 2011, 115, 5913–
5922; b) M. R. Berber, H. Mori, I. H. Hafez, K. Minagawa, M.
Tanaka, T. Niidome, Y. Katayama, A. Maruyama, T. Hirano, Y.
Maeda, T. Mori, J. Phys. Chem. B 2010, 114, 7784–7790.
[29] a) R. Freitag, F. Garret-Flaudy, Langmuir 2002, 18, 3434–3440;
b) K. Van Durme, H. Rahier, B. V. Mele, Macromolecules 2005, 38,
10155–10163; c) Y. J. Zhang, S. Furyk, D. E. Bergbreiter, P. S.
Cremer, J. Am. Chem. Soc. 2005, 127, 14505–14510.
[30] a) Z. W. Wang, H. J. Lai, P. Y. Wu, Soft Matter 2012, 8, 11644–
11653; b) H. Mori, M. Yahagi, T. Endo, Macromolecules 2009, 42,
8082–8092.
[31] Q. H. Wu, C. Wang, D. Zhang, X. M. Song, D. L. Liu, L. P. Wang,
G. L. Zhang, React. Funct. Polym. 2012, 72, 372–377.
[32] E. R. Blout, R. H. Karlson, J. Am. Chem. Soc. 1956, 78, 941–946.
[33] W. H. Daly, D. Pochꢃ, Tetrahedron Lett. 1988, 29, 5859–5862.
[34] Q. H. Wu, C. Wang, D. Zhang, X. M. Song, F. Verpoort, G. L.
Zhang, React. Funct. Polym. 2011, 71, 980–984.
Received: May 19, 2014
Published online: August 21, 2014
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