Thermo- and pH-responsive polymer derived from methacrylamide and aspartic acid

Article abstract of DOI:10.1021/ma1015227

A new type of thermo- and pH-responsive homopolymer was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. A novel functional methacrylamide monomer bearing α-aspartic acid derivative in the side chain, i.e., N-metha

Full text of DOI:10.1021/ma1015227

Macromolecules 2010, 43, 8101–8108 8101
DOI: 10.1021/ma1015227
Thermo- and pH-Responsive Polymer Derived from Methacrylamide
and Aspartic Acid
Chunhui Luo, Yu Liu, and Zhibo Li*
Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100190, China
Received July 8, 2010; Revised Manuscript Received August 28, 2010
ABSTRACT: A new type of thermo- and pH-responsive homopolymer was synthesized using reversible
addition-fragmentation chain transfer (RAFT) polymerization. A novel functional methacrylamide mono-
mer bearing R-aspartic acid derivative in the side chain, i.e., N-methacryloyl-L-β-isopropylasparagine benzyl
ester (MA-iAsn-OBen), was first synthesized. Three poly(N-methacryloyl-L-β-isopropylasparagine benzyl ester)s
[poly(MA-iAsn-OBen)] with controllable molecular weight and narrow molecular weight distribution were
then prepared using RAFT polymerization. Selective removal of benzyl groups produced poly(N-methacryloyl-
L-β-isopropylasparagine) [poly(MA-iAsn-OH)], which displayed a reversible lower critical solution tem-
perature (LCST) in water. In addition, the LCST can be adjusted from 29 to 60 °C by changing solution
pH values, salt concentrations, and polymer molecular weights. Using macromolecular chain transfer agent
and RAFT polymerization, we prepared well-defined amphiphilic mPEG45-b-poly(MA-iAsn-OBen)53 diblock
copolymer and double hydrophilic block copolymer [mPEG45-b-poly(MA-iAsn-OH)53]. The latter showed
a thermo-induced self-assembly in water due to collapse of poly(MA-iAsn-OH) segment. We found that
mPEG45-b-poly(MA-iAsn-OH)53 was soluble at room temperature while formed vesicles above the LCST of
poly(MA-iAsn-OH)53.
Introduction
amino acids in the side chains for many years.27-29,35-38 For
example, they developed novel thermo-responsive (co)polymers
Environmentally responsive polymers are termed “smart” mate-
rials, which can respond to external stimuli, such as temperature,
pH, multivalent ions, ionic strength, and biomolecules, to induce
substantial changes of material properties. These smart materials
have found extensive applications in tissue engineering, drug
delivery, bioseparations, biosensors, and nanomedicines.1-5 In
particular, polymers that can respond to multiple external stimuli
are of particular interest to constructing high-ordering and
hierarchical supramolecular structures. Generally, different poly-
meric units that have distinct responsive moieties were incorpo-
rated into one copolymer system, which will display multiple
responsive mechanisms. Among them, a widely studied dual-
responsive system is thermo- and pH-responsive copolymers.6-12
The critical responsive pH and temperature can be adjusted
througth variation of copolymer constitutions, compositions, and
architectures, etc. Such a dual responsive mechanism certainly
can offer additional control over resulting supramolecular assembly
structures as well as their functionalities.
Recently, much attention has been devoted to preparing poly-
mers containing amino acids due to their biocompatibilities
as well as their feasibility to create high-ordered structures.13-19
Although these hybrid copolymers can be prepared via various
methods,20,21 great effort has been paid to the controlled/living
radical polymerization (CRP).15,19,22-32 The primary reason is
that CRP has high tolerance for various functional groups and
precise control over polymer composition and architecture.33,34
For example, van Hest et al. synthesized a methacrylate- functio-
nalized monomer bearing the short peptide sequence (VPGVG)
in the side chain using ATRP and found that these polymers had
dual pH- and thermo-sensitivity.15,23,24 The Endo group have
studied the synthesis and properties of polymers containing different
containing a N-acryloyl-L-proline methyl ester (A-Pro-OMe)
unit.27,35-37 They first reported RAFT polymerization of A-Pro-
OMe and obtained well-defined thermo-responsive polymers,
whose phase separation temperature can be tuned simply by copoly-
merization with hydrophilic monomers such as N,N-dimethyl-
acrylamide (DMA)27 and N-acryloyl- -proline (A-Pro-OH).36
L
Later, they investigated structures and chiroptical properties of
thermo-responsive block copolymers containing
-proline moieties.35
L
Most recently, Mori and co-workers reported dual-stimuli respon-
sive block copolymers consisting of a thermo-responsive segment
[poly(A-Pro-OMe)] and a weak anionic polyelectrolyte [poly(A-
Pro-OH)].36 In particular, they found that poly(A-Pro-OMe)-b-
poly(N-acryloyl-4-trans-hydroxy-L-proline) displayed lower critical
solution temperature (LCST) and upper critical solution tempe-
rature (UCST).37 Also, McCormick and co-workers studied the
self-assembly of multistimuli responsive block copolymers con-
taining poly(N-acryloylalanine) and poly(N-acryloylvaline) using
RAFT polymerization.25,26 The O’Reilly group studied the for-
mation of micelle from amphiphilic block copolymers containing
amino acids as side chains and self-assembly of amphiphilic chiral
poly(amino acid) star polymers.17,18 Moreover, Adams and Young
reported formation of vesicle from poly(ethylene oxide)-b-poly-
(methacryloyl side-chain peptides).14 However, to the best of our
knowledge, only two groups of amino acids related poly(meth)acryl
derivatives exhibit thermo-responsive properties. One is elastin-
based (co)polymers, and the other is proline-based (co)polymers.
Therefore, we are interested in developing new types of functional
polymers containing R-amino acids as side chains and hope to
expand the natural R-amino acids as functional groups to cons-
truct dual-stimuli responsive polymeric materials.
Herein, we attempted to incorporate thermo-responsive and
pH-sensitive unit into a single molecule and utilized CRP to
*Corresponding author. E-mail: zbli@iccas.ac.cn.
r 2010 American Chemical Society
Published on Web 09/17/2010
pubs.acs.org/Macromolecules

8102 Macromolecules, Vol. 43, No. 19, 2010
Luo et al.
prepare well-defined (co)polymers. We first designed a new vinyl
solution once. The organic layer was dried over anhydrous MgSO4,
and the solvent was removed via rotary evaporatin to give crude
product. The crude product was subsequently purified by recrys-
tallization three times using THF/petroleum ether. The isolated
yield was 44%. 1H NMR (400 MHz, CDCl3,TMS):δ=7.35(C6H5,
5H, s), 7.22 (NHCHCH2, 1H, s), 6.41 (NHCH(CH3)2, 1H, s), 5.78
monomer, namely N-methacryloyl-
zyl ester (MA-iAsn-OBen). The side chain of this monomer was
-aspartic acid derivative, in which the β-carboxyl group was
L-β-isopropylasparagine ben-
L
converted into isopropyl amide group to mimic the structure
of N-isopropylacrylamide for the purpose of thermo-responsive
property. To reach a good control over the balance between
hydrophobicity and hydrophilicity, we kept the -COOH group
in order to have pH sensitivity. Then we employed RAFT
polymerization followed by selective removal of benzyl ester
groups to obtain target polymers. We also investigated the
aqueous solution properties of poly(MA-iAsn-OH) homopoly-
mers and double hydrophilic mPEG45-b-poly(MA-iAsn-OH)53
diblock copolymer.
0
and 5.38 (CHaHa dCCH3, 1H, s and 1H, s), 5.21 (COOCH2Ph,
2H, q), 4.84 (NHCHCH2, 1H, m), 4.01 (NHCH(CH3)2, 1H, m),
2.62-3.06 (CHCH2CO, 2H, m), 1.97 (CH2dCCH3, 3H, s), 1.12
(NHCH(CH3)2, 6H, m). 13C NMR (400 MHz, CDCl3): δ=172.01
(COOCH2), 169.31 (CONHCHCH2), 168.14 (CONHCH(CH3)2),
139.10 (CH2dCCH3), 135.46 (ArC-1), 128.68 (ArC-2, ArC-3, and
ArC-4), 120.92 (CH2dCCH3),66.97(CH2Ar), 49.51(NHCHCH2),
41.76 (NHCH(CH3)2),36.07(CHCH2CONH), 22.57 (CH(CH3)2),
18.47 (CH2dCCH3). High-resolution mass spectrum (HRMS):
m/z calcd for C18H24N2O4 [M þ H]þ 333.1814; found 333.1809.
Anal. Calcd for C18H24N2O4: C, 65.04; H, 7.28; N, 8.43. Found: C,
65.07; H, 7.25; N, 8.32.
Experimental Section
Materials. Boc-L-Asp-OBen, dicyclohexylcarbodiimide (DCC),
and 4-(dimethylamino)pyridine (DMAP) were purchased from GL
Biochem (Shanghai) Ltd. and used as received. Isopropylamine
(99%) and monomethoxypoly(ethylene glycol) (mPEG45, Mn =
2000 g/mol) were obtained from Aldrich. All organic solvents were
purchased from Beijing Chemical Co. Other reagents were pur-
chased from Aladdin and used as received unless otherwise stated.
2,20-Azobis(isobutyronitrile) (AIBN) was purified by recrystalliza-
tion from methanol. Dichloromethane (DCM), tetrahydrofuran
(THF), and n-hexane were dried by purging with nitrogen and
passing through alumina columns prior to use. Deionized water was
obtained from a Millipore Milli-Q purification unit. 2-Cyanoprop-
2-yl(4-fluoro)dithiobenzoate (CPFDB),39 4-cyanopentanoic acid
dithiobenzoate (CPADB),40,41 and mPEG45-macro-CTA42 were
synthesized according to reported procedures.
Synthesis of N-Methacryloyl-β-isopropylasparagin Benzyl Ester
(MA-iAsn-OBen). a. Synthesis of Boc-L-β-isopropylasparagine-
OBen. Boc-L-Asp-OBen (30 mmol, 10 g), DMAP (3 mmol, 0.36 g),
and DCC (30 mmol, 6.18 g) were dissolved in 100 mL of anhydrous
THF with magnetic stirring bar. While stirring vigorously, iso-
propylamine (30 mmol, 1.77 g) was added dropwise and then
stirred for 24 h at RT. Dicyclohexyurea (DCU) was filtered off.
Organic solvent was removed by rotary evaporation to give white
crude product, which was purified by recrystallization using
acetone/petroleum ether. Boc-L-β-isopropylasparagine-OBen was
obtained in 80% yield. 1H NMR (400 MHz, CDCl3, TMS): δ =
7.35 (C6H5, 5H, s), 6.25 (NHCH(CH3)2, 1H, s), 5.65 (NHCHCH2,
1H, s), 5.21 (COOCH2Ph, 2H, q), 4.84 (NHCHCH2, 1H, m), 4.01
(NHCH(CH3)2, 1H, m), 2.62-3.06 (CHCH2CO, 2H, m), 1.46
(OC(O)(CH3)3, 9H, s), 1.12 (NHCH(CH3)2, 6H, m).
b. Synthesis of L-β-Isopropylasparagine-OBen. Boc-L-β-iso-
propylasparagine-OBen (24 mmol, 8.16 g) was dissolved in 36 mL
of anhydrous DCM at 0 °C followed with addition of TFA (0.1 mol,
9 mL). The mixture was slowly warmed up to RT and stirred
for 2 h. An additional 100 mL of DCM was then added. The
solution was washed with saturated NaHCO3 and NaCl solu-
tion twice each. The organic layer was dried over anhydrous
MgSO4, and the solvent was then removed by rotary evapora-
tion. The isolated yield was 95%. 1H NMR (400 MHz, CDCl3,
TMS): δ=7.78 (NH2CHCH2, 2H, d), 7.35 (C6H5, 5H, s), 6.25
(NHCH(CH3)2, 1H, s), 5.21 (COOCH2Ph, 2H, q), 4.01 (NHCH-
(CH3)2, 1H, m), 3.85 (NH2CHCH2, 1H, m), 2.62-3.06 (CHCH2-
CO, 2H, m), 1.12 (NHCH(CH3)2, 6H, m).
c. Synthesis of N-Methacryloyl-L-β-isopropylasparagine Benzyl
Ester (MA-iAsn-OBen). L-β-Isopropylasparagine-OBen (24 mmol,
6.33 g) and triethylamine (30 mmol, 3.03 g) were dissolved in 60 mL
of anhydrous DCM, and the solution was cooled to 0 °C using
an ice bath. Methacryloyl chloride (24 mmol, 2.51 g) dissolved in
10 mL of anhydrous DCM was added dropwise over 15 min. The
mixture was slowly warmed up to RT and stirred for 24 h. The
precipitated ammonium salt was removed by filtration. The DCM
solution was subsequently washed with 1 M HCl and satu-
rated NaHCO3 solution twice each followed with saturated NaCl
Synthesis of Poly(MA-iAsn-OBen) Homopolymers. All poly-
merizations of MA-iAsn-OBen were performed in a degassed
sealed tube using CPFDB as the chain transfer agent (CTA) and
AIBN as the initiator. The initial monomer-to-CTA ratio
([M]0/[CTA]0) was varied between 50 and 500 while the initial
CTA-to-initiator ratio ([CTA]0/[I]0) was held constant at 2:1. A
representative example for RAFT polymerization of MA-iAsn-
OBen monomer was as follows: MA-iAsn-OBen (332 mg, 1 mmol),
CPFDB (2.39 mg, 0.01 mmol), AIBN (0.82 mg, 0.005 mmol),
and methanol (1 mL) were charged into a dry ampule. The
solution was then deoxygenated using three freeze-evacuate-
thaw cycles before the ampule was flame-sealed under vacuum.
The ampule was then immersed into oil bath at 60 °C for 24 h.
The polymerization was stopped by rapid cooling and exposure
solution to air followed with methanol dilution. The solution
was sampled for 1H NMR and GPC measurements. The remaining
product was precipitated using excess ethyl ether, and the preci-
pitate was collected and dried in a vacuum. Conversion was
assessed using 1H NMR based on eq 1, where I5.78 and I5.38 are
the integral of vinyl protons of CH2dCCH3 in MA-iAsn-OBen
monomer, and I5.21 is the integral of methylene protons reso-
nance of COOCH2Ph in poly(MA-iAsn-OBen) polymer and
MA-iAsn-OBen monomer. The conversion was 63%, determined
from 1H NMR.
conversion ¼ ðI5:21 - I5:78 - I5:38Þ=I5:21
ð1Þ
Synthesis of Block Copolymer. mPEG45-CTA was used as chain
transfer agent for preparing diblock copolymer of mPEG45-b-
poly(MA-iAsn-OBen). The polymerization was carried out in
methanol with an initial monomer concentration of 1.0 M at 60 °C
with AIBN as the initiator. The [macroCTA]0:[AIBN]0 ratio was
maintained at 2, and [M]0:[mPEG45-CTA] ratio was 50. The proce-
dure of making block copolymer was similar to homopolymer
described above except that mPEG45-CTA was used. The obtained
Mn from GPC/LS was 17.2 kDa with PDI = 1.11. The DP of
1
poly(MA-iAsn-OBen) was 53 determined from H NMR using
known mPEG45 as reference.
Removal of Benzyl Ester. The conversion of poly(MA-iAsn-
OBen) to poly(MA-iAsn-OH) was carried out by the standard
method following literature procedure.43-45 100 mg of poly-
(MA-iAsn-OBen) and a trace of ascorbic acid were dissolved in
1 mL of TFA at 0 °C, and then 0.5 mL of HBr/acetic acid (33%)
solution was added. After stirring at RT for 2 h, 20 mL of diethyl
ether was added. The precipitated polymer was dissolved in
water and then dialyzed again in water for 72 h. After being
lyophilized, the target homopolymer, poly(MA-iAsn-OH), was
obtained in 85% yield. A similar deprotection procedure was
applied for copolymers. Success of deprotection was confirmed
1
from H NMR characterization and GPC measurement. The
GPC/LS measurement of mPEG45-b-poly(MA-iAsn-OH)53 was
carried out after silylation of the free carboxylic groups with
trimethylchlorosilane46 (Mn =15.1 kDa with PDI =1.14).

Article
Preparation of Copolymer Solutions. mPEG45-b-poly(MA-iAsn-
Macromolecules, Vol. 43, No. 19, 2010 8103
OH)53 diblock was dissolved directly in DI water (c=1 mg/mL).
The solution pH was adjusted to desired values using 0.1 M HCl or
NaOH for LCST, DLS, and cryo-TEM measurements.
Characterization. 1HNMRand13C NMR spectra were recorded
on a Bruker AV400 FT-NMR spectrometer. High-resolution mass
spectrometry (HRMS) was recorded on a Bruker Daltonics Inc.
APEX II FT-ICRMS spectrometer. Elemental analysis was carried
out using a Flash EA 1112 analyzer. CD spectra were recorded on
an Applied Photophysics Chirascan CD spectrometer. Tandem gel
permeation chromatography/light scattering (GPC/LS) was done
at 50 °C using an SSI pump connected to Wyatt Optilab DSP and
Wyatt DAWN EOS light scattering detectors with 0.02 M LiBr in
DMF as eluent at flow rate of 1.0 mL/min. Dynamic light scattering
(DLS) measurements were performed using LLS spectrometer
(ALV/DLS/SLS-5322F) equipped with a multi-τ digital time cor-
relator (ALV5000) and a He-Ne Laser (λ=632.8 nm) at an angle
of 90°. All solutions were filtered through a 0.45 μm PVDF filter
prior to measurements. The LCSTs were measured by monitoring
the transmittance of a 500 nm light beam through a quartz sample
cell at concentration of 1 mg/mL on a TU-1901 (Beijing Purkinje
General Instrument Co. Ltd.) UV-vis spectrophotometer equip-
pedwith a temperature controller system (Peltier Temperature
Controller-2). The solution was heated or cooled at rate of 1 °C/min
between 20 and 70 °C. LCST was determined at 50% transmittance.9
For cryogenic transmission electron microscopy (cryo-TEM) char-
acterization, samples were prepared in a controlled environment
vitrification system (CEVS) at preset temperature. Block copoly-
mer solution was annealed in the CEVS system for 10 min at each
temperature prior to sample preparation. Typically, a 5 μL sample
solution was loaded onto a carbon-supported lacey TEM grid,
which was held by tweezers. The excess solution was blotted with a
piece of filter paper, resulting in the formation of thin films
suspended the mesh holes, and the samples were quickly plunged
into a reservoir of liquid ethane (cooled by liquid nitrogen) at its
melting temperature. The vitrified samples were then stored in
liquid nitrogen until they were transferred to a cryogenic sample
holder (Gatan 626) and examined using a JEM 2200FS TEM
(200 keV) at about -174 °C. The phase contrast was enhanced by
underfocus. The images were recorded on a Gatan multiscan CCD
and processed with Digital Micrograph.
Figure 1. (a) 1H NMR and (b) 13C NMR spectra of N-methacryloyl-
β-isopropylasparagine benzyl ester (MA-iAsn-OBen) in CDCl3.
Results and Discussion
Synthesis of MA-iAsn-OBen Monomer. The synthesis of poly-
merizable MA-iAsn-OBen monomer is outlined in Scheme 1.
An orthogonal protected L-aspartic acid (Boc-L-Asp-OBen) as
precursor was first coupled with isopropylamine to give Boc-
L-β-isopropylasparagine-OBen. Selective removal of Boc-
protective group using TFA produced L-β-isopropylasparagine-
OBen, which subsequently reacted with methacryl chloride to
produce target MA-iAsn-OBen monomer, whose structure
was verified using 1H NMR, 13C NMR (Figure 1), elemental
analysis, and HRMS. 1H NMR characterization showed that
the integral ratio of proton resonance of Ia:Ib:Ic:Id:Ie:If:Ig:Ih:Ii:Ij
is equal to 2:3:2:5:1:2:1:6:1:1 (Figure 1a), which agreed well with
its corresponding structure. The 13C NMR spectrum shown in
Figure 1b displayed the expected resonance and further con-
firmed successful synthesis of target monomer. HRMS and
elemental analysis results were consistent well with theoretical
values.
Scheme 1. Synthetic Route to Poly(N-methacryloyl-
L-β-isopropylasparagine) [Poly(MA-iAsn-OH)]
RAFT Polymerization of MA-iAsn-OBen. For understand-
ing the relationship between physical properties and polymer
structures, it is always desirable to make (co)polymers with
controlled molecular weights and narrow molecular weight
distribution (MWD). Considering the target MA-iAsn-OBen
monomer containing amino acid unit and amide bond, we
employed RAFT polymerization, which has been shown to have
good control over such types of monomers.26,27 Previous research
demonstrated that methanol and CPFDB were efficient for
RAFT polymerization of methacrylate-functionalized mono-
mers.39,47 Hence, we applied CPFDB as CTA to polymerize
MA-iAsn-OBen monomer in methanol and prepared three
poly(MA-iAsn-OBen) homopolymers with narrow MWD
(Table S1). The obtained poly(MA-iAsn-OBen) homopolymers
were found to be soluble in most organic solvents such as THF,
DMSO, DMF, and methanol, etc., but insoluble in water.

8104 Macromolecules, Vol. 43, No. 19, 2010
Luo et al.
Table 1. LCST of 0.1 wt % Aqueous Solutions of
Poly(MA-iAsn-OH) Homopolymers at Different pH Values
LCST/°C
samples
pH 1
pH 2
pH 3
38
pH 4
53
poly(MA-iAsn-OH)295
poly(MA-iAsn-OH)50
poly(MA-iAsn-OH)30
turbid
41
60
29
49
narrow (Mw/Mn = 1.11-1.24). Apparently, RAFT poly-
merization of MA-iAsn-OBen showed controlled polymer-
ization characteristic, which allowed a good control over
molecular weight of poly(MA-iAsn-OBen) for subsequent
solution phase behavior studies.
1
Figure 3b shows the H NMR of poly(MA-iAsn-OBen).
The characteristic resonances of poly(MA-iAsn-OBen) are
easily identified, and their integral ratio of Ia:I(bþh):Ic:Id:Ie:If:
Ig is close to 2:9:2:5:1:2:1 (Figure 3b), in good agreement with
its corresponding chemical structure. Selective removal of
benzyl ester from poly(MA-iAsn-OBen) produced target
Figure 2. GPC traces of poly(MA-iAsn-OBen) at different [M]0/
[CPFDB]0 ratios.
1
poly(MA-iAsn-OH) (Scheme 1), as the H NMR spectrum
shown in Figure 3a clearly verified the successful deprotec-
1
tion of the benzyl group. Compared to the H NMR spec-
trum of poly(MA-iAsn-OBen) (Figure 3b), resonances of
aromatic (δ=7.3 ppm) and methylene (δ=5.2 ppm) protons
from benzyl group disappeared while other characteristic
resonances as well as the integral ratio remained intact after
deprotection. Furthermore, the 1H NMR spectrum did not
display unknown resonances, suggesting that removal of
benzyl group did not introduce unexpected structures. The
target poly(MA-iAsn-OH) homopolymers were found solu-
ble in water at neutral pH and displayed a reversible LCST
behaviors in water at low pH, as discussed below.
We employed circular dichroism (CD) spectroscopy to
assess the optical activity of the polymer before and after
deprotection in methanol at RT (Figure S1). Poly(MA-iAsn-
OBen)50 exhibits a negative CD signal at 206 nm and a
positive CD signal at 234 nm with intensities of about
-32 800 and 2400 deg cm2 dmol-1, respectively. This result
was similar to poly(A-Pro-OMe) systems reported by Endo
and co-workers.35,36 After deprotection, the poly(MA-iAsn-
OH)50 exhibits a negative CD signal at 197 nm and a positive
CD signal at 224 nm, and the intensities were changed to
about 31 000 and 6000 deg cm2 dmol-1, respectively. These
results were consistent with similar systems containing
-COOH in their side chain.48,49 Typically, the peak around
200 nm was attributed to the π2 f π* transition or the n f σ
transition of the amide group, while the signals around 207
and 230 nm were attributed to the n f π* transition of the
carboxyl chromophore and π1 f π* transition of the amide
chromophore, respectively.49
Phase Separation of Poly(MA-iAsn-OH) in Aqueous Solu-
tion. After successfully preparing poly(MA-iAsn-OH), we
started to investigate their physical properties in water. Three
poly(MA-iAsn-OH) homopolymers with DP being 30, 50,
and 295 were used for this study (Table 1). The samples were
designated as poly(MA-iAsn-OH)n with n representing the
degree of polymerization (DP).
Initial experiments showed that these poly(MA-iAsn-OH)
homopolymers were soluble at neutral pH without measur-
able LCST behaviors between room temperature and 100 °C.
The reason we assumed was due to the ionization of -COOH
groups, which are weak acid with pKa around 4.2.26 At pH=7,
almost all -COOH groups will dissociate into -COO-
groups. Considering the contribution of ionic groups toward
poly(MA-iAsn-OH) solubility, we decided to decrease the
pH values in order to protonate -COO- group. We aim to
Figure 3. 1H NMR spectra of (a) poly(N-methacryloyl-
-β-isopropyl-
asparagine)50 [poly(MA-iAsn-OH)50] and (b) poly(N-methacryloyl-
β-isopropylasparagine benzyl ester)50 [poly(MA-iAsn-OBen)50] in
CD3OD. The peaks marked with asterisks indicate solvents.
L
Figure 2 shows the GPC traces of the poly(MA-iAsn-
OBen) prepared at different [monomer]0/[CTA]0 ratios with
[CTA]0:[AIBN]0 = 2. For these three polymerizations, the
conversions were around 60% (Table S1). The molecular
weight of poly(MA-iAsn-OBen) increased monotonically with
[monomer]0/[CTA]0 ratio (Figure S2), and the MWD remained

Article
Macromolecules, Vol. 43, No. 19, 2010 8105
compensate the enhanced hydrophilicity from ionic -COO-
groups along increase of solution pH. Figure 4b also shows
the cooling histogram of transmittance vs temperature,
suggesting reversible soluble-insoluble phase transition for
poly(MA-iAsn-OH) system. The LCST determined from
cooling was about 1 deg lower than that determined from
the heating ramp. The hysteresis between heating and cool-
ing cycles most likely arose from the inter- and intrachain
hydrogen bonding at insoluble status as suggested in litera-
ture reports.51,52
Our experiments showed that the LCST of poly(MA-iAsn-
OH) decreased with increasing polymer molecular weight
(Figure 4c), which is consistent with literature reports.23,47,53
For the example of pH=1, poly(MA-iAsn-OH)295 has LCST
below room temperature, while poly(MA-iAsn-OH)30 and
poly(MA-iAsn-OH)50 has LCST around 60 and 41 °C,
respectively. This trend was similar to that of PNIPAM
system (from 32 to 70 °C).54 Generally, the molecular weight
dependence of LCST could arise from several factors includ-
ing polymer-solvent interaction, intramolecular interactions,
and end-group contributions.53 In particular, the end-group
effect was significant for the low molecular weight samples
and became less remarkable with increase of polymer chain
length.54,55 Apparently, the end group from CTA would have
significant influence on the LCST of poly(MA-iAsn-OH), as
expected. However, we could not quantify the contribution
of end group toward LCST given the available data. Mean-
while, we also note that the LCST variation range was just
a few degrees for PNIPAM having same end group but
different molecular weight. Here, the LCST for poly(MA-
iAsn-OH) spanned from at least 25 to 60 °C when DP changed
from 295 to 30. Therefore, another possible contribution we
assume might be the enhanced intramolecular hydrogen
bonding between polar-NHCO- and -COOH groups when
the poly(MA-iAsn-OH) chain length increases.
We also found that solution ionic strength can also affect
the LCST of poly(MA-iAsn-OH) aqueous solutions (Figure 4d).
Here, we chose NaCl as the additive salt for our studies. For
1 mg/mL aqueous solution of poly(MA-iAsn-OH)50, addition
of different amounts of NaCl progressively lowered its LCST
from 49 without NaCl to 46, 42, and 28 °C at [NaCl] = 0.5,
1.0, and 1.5 M, respectively. The underlying reason we believed
was due to ionic nature of -COOH groups. Since poly(MA-
iAsn-OH) is a weak polyelectrolyte, the ionic strength can
affect the degree of ionization, and consequently polymer
solubility provided other parameters were identical. There-
fore, addition of NaCl will decrease poly(MA-iAsn-OH)
solubility in water, and corresponding LCST will decrease
accordingly (Figure 4d).23,36
Figure 4. Temperature dependence of the transmittance at 500 nm of
1 mg/mL poly(MA-iAsn-OH) aqueous solutions: (a) poly(MA-iAsn-
OH)295 at different pH, (b) poly(MA-iAsn-OH)50 at different pH,
(c) poly(MA-iAsn-OH) with different DP at pH = 1, and (d) poly(MA-
iAsn-OH)50 solution at different NaCl concentrations with pH = 2.
find hydrophilic and hydrophobic balance among polymer
backbone, isopropylamide, and -COOH groups and ulti-
mately realized a soluble-insoluble transition upon increase
of temperature. Interestingly, poly(MA-iAsn-OH) homopo-
lymers started to display a reversible soluble-insoluble
phase transition with increase of temperature when pH < 5
(Figure4). WealsofoundthattheLCSTof poly(MA-iAsn-OH)
varies with molecular weight and solution ionic strength. We
thus systematically explored the roles of polymer molecular
weight, pH value, and ion strength on polymers LCST beha-
viors in water as discussed below.23,36,50
Figure 4a shows the solution turbidity of poly(MA-iAsn-
OH)295 aqueous solution (1 mg/mL) versus temperature at
different pH. When pH = 1, the LCST of poly(MA-iAsn-
OH)295 was not obtainable as we did not have cooling unit in
the turbidity apparatus. Its LCST seemed below room tem-
perature (∼25 °C). When pH = 2, poly(MA-iAsn-OH)295
showed a soluble-insoluble transition at 29 °C. The phase
transition started at 25 °C with 90% transmittance and
ended at 32 °C with 5% transmittance, which gave a phase
transition range about 7 deg. When the pH was increased
to 3 and 4, poly(MA-iAsn-OH)295 had its LCST increased
to 38 and 53 °C, respectively (Figure 4a). Further increas-
ing solution pH to 5, poly(MA-iAsn-OH)295 did not show
distinguishable LCST. In contrast, poly(MA-iAsn-OH)50 at
pH = 1 had LCST of 41 °C (Figure 4b), which was much
higher than that of poly(MA-iAsn-OH)295 under the same
conditions. When pH=2, poly(MA-iAsn-OH)50 underwent
a phase transition around 49 °C, in contrast to 29 °C for
poly(MA-iAsn-OH)295. At pH = 3, poly(MA-iAsn-OH)50
did not display a sharp LCST transition, and its solution still
had 70% transmittance even at 70 °C (Figure 4b). Appar-
ently, the LCST of poly(MA-iAsn-OH) strongly depended
on solution pH. We attributed this to the presence of ion-
izable -COOH groups, which endowed ionic nature to poly-
(MA-iAsn-OH). As a result, the LCST of poly(MA-iAsn-OH)
is primarily determined by the balance between thermo-
dependent hydrophobic unit and ionizable -COOH groups.
The higher the solution pH, the more ionic -COO- groups,
which improve the polymer solubility.23,26,36 As a result,
increase of phase transition temperature was required to
It needs pointing out that the dual-responsive mechanism
of poly(MA-iAsn-OH) arose from a single monomer bearing
two distinct functional groups, so copolymerization was not
necessary to obtain dual-stimuli-responsive property. An addi-
tional benefit was that we can rather easily prepare multiple-
responsive polymer via copolymerization with other specific
monomers.
Synthesis of mPEG45-b-poly(MA-iAsn-OH)53 Copolymer.
We have demonstrated that poly(MA-iAsn-OH) homopo-
lymer has dual thermo- and pH-responsive properties
in water. We thought about using its stimuli mechanism
to make responsive block copolymers, from which we
can construct intelligent drug delivery systems. We chose
mPEG45-CTA to perform RAFT polymerization of MA-
iAsn-OBen and obtained mPEG45-b-poly(MA-iAsn-OBen).
The [monomer]0/[mPEG45-CTA]0 is 50, and conversion of
1
monomer was >95% determined by H NMR right after
polymerization.

8106 Macromolecules, Vol. 43, No. 19, 2010
Luo et al.
Figure 7. Apparent size distribution of aggregates formed from 0.1 wt %
mPEG45-b-poly(MA-iAsn-OH)53 aqueous solution at (a) 60 °C with
pH = 2 and (b) 50 °C with pH = 1. The scattering angle is 90°.
Figure 5. GPC traces of mPEG45-CTA (solid) and mPEG45-b-poly-
(MA-iAsn-OBen)53 (dashed) diblock copolymer.
we can estimate that purity of mPEG45-CTA was 95%
(containing 5% inactive mPEG45), which also explained
the small shoulder observed in GPC trace (Figure 5). More-
over, the GPC/LS measurement gave Mn of diblock being
17.2 kDa, from which we can estimate the DP of poly(MA-iAsn-
OBen) block was 46, which was smaller than NMR results.
The success of deprotection was confirmed from 1H NMR
as the resonances of benzyl group completely disappeared
while other characteristic resonances as well as the integral
ratio remained intact (Figure 6). We attempted to use hydro-
genation to remove benzyl group, but could not obtain 100%
deprotection. So we adopted a more aggressive HBr/acetic
acid system to deprotect benzyl group.43,45 Although the
ester linkage between mPEG45 and poly(MA-iAsn-OBen)53
is subject to acid catalyzed cleavage, we tried to control
reaction conditions in order to minimize the possible side
reaction. Our result showed that mPEG45-b-poly(MA-iAsn-
OH)53 did not have substantial chain cleavage supported
from 1H NMR and GPC (Figure S3). Above all, the selective
deprotection of benzyl group was successful to produce double
hydrophilic mPEG45-b-poly(MA-iAsn-OH)53 diblock, which
is soluble in water within all pH ranges at room temperature.
Since poly(MA-iAsn-OH)53 is thermo- and pH-responsive
block, we expect it will form supramolecular aggregates at
elevated temperature and low pH value. We then started to
investigate the thermo-induced aqueous self-assembly of
mPEG45-b-poly(MA- iAsn-OH)53.
Solution Behaviors of mPEG45-b-poly(MA-iAsn-OH)53
Diblock. Because of the thermo-responsive characteristic of
poly(MA-iAsn-OH), mPEG45-b-poly(MA-iAsn-OH)53 will
undergo a thermo-induced transition from double hydro-
philic diblock to amphiphlic diblock upon temperature
increase. We knew that poly(MA-iAsn-OH) displayed LCST
behaviors only when most -COOH groups were proto-
nated, so we mainly investigated the thermo-induced aque-
ous self-assembly of mPEG45-b-poly(MA-iAsn-OH)53 diblock
at pH=1 and 2. For 1 mg/mL aqueous solution of mPEG45-
b-poly(MA-iAsn-OH)53 diblock, visual observation showed
the solution became cloudy at elevated temperature when
pH =1 or 2.
We employed dynamic light scattering (DLS) to monitor
scattering intensity and apparent aggregate size dependence
on temperature (Figure S4a). The mPEG45-b-poly(MA-
iAsn-OH)53 aqueous solution displayed LCST around 40 °C
at pH=1 and 48 °C at pH =2, consistent well with corres-
ponding homopolymer with DP = 50 (Figure 4). Also, the
soluble-insoluble transition was reversible as indicated from
DLS measurements (Figure S4a). In addition to scattering
Figure 6. 1H NMR spectra of (a) mPEG45-b-poly(MA-iAsn-OH)53
and (b) mPEG45-b-poly(MA-iAsn-OBen)53 diblock copolymer in CD3OD.
The peaks marked with asterisks indicate solvents.
Figure 5 compares the GPC traces of mPEG45-CTA and
mPEG45-b-poly(MA-iAsn-OBen). Compared to mPEG45-
CTA, mPEG45-b-poly(MA-iAsn-OBen) diblock had sub-
stantial decrease of elution volume, indicating a successful
extension of polymer chain from mPEG45-CTA. Although
the GPC trace of mPEG45-b-poly(MA-iAsn-OBen) diblock
had a small shoulder peak at the low molecular region, we
believed the shoulder was caused by inactive mPEG45-CTA.
Also, its contribution to diblock would be minimal as sup-
ported by the relative narrow molecular weight distribu-
tion of diblock (PDI=1.11). From 1H NMR (Figure 6b) of
mPEG45-b-poly(MA-iAsn-OBen) diblock, the DP of poly-
(MA-iAsn-OBen) block was calculated to be 53 given Mn of
mPEG45-CTA. The diblock will be designated as mPEG45-
b-poly(MA-iAsn-OBen)53 for remaining text. On the basis of
the monomer conversion and DP of poly(MA-iAsn-OBen),

Article
Macromolecules, Vol. 43, No. 19, 2010 8107
aspartic acid via RAFT polymerization. We found that the LCST
of poly(MA-iAsn-OH) in water increases with solution pH when
pH < 5. Increase polymer chain length or solution salt concen-
tration will decrease LCST for dilute poly(MA-iAsn-OH) aqu-
eous solution. Using mPEG45-CTA as macro-CTA, we made
amphiphilic mPEG45-b-poly(MA-iAsn-OBen)53, which was con-
verted into double hydrophilic mPEG45-b-poly(MA-iAsn-OH)53
diblock copolymer. The former formed vesicles in water, while
the latter underwent a transition from polymer chain to vesicular
assemblies upon increase of solution temperature. This new type
of stimuli-responsive polymer prepared from a single monomer
might be useful to prepare multiresponsive polymers for drug
delivery and biomedical sensors applications.
Acknowledgment. This work was supported by the National
Natural Science Foundation of China (20974112, 50821062). We
thank Dr. Yi Shi for the help in RAFT agent synthesis and useful
discussions.
Supporting Information Available: CD spectra, additional
GPC, DLS, and cryo-TEM data, and NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
Figure 8. Cryo-TEM micrographs of assemblies formed from 0.1 wt %
mPEG45-b-poly(MA-iAsn-OH)53 aqueous solution at (a, b) 50 °C with
pH = 1 and (c, d) 60 °C with pH = 2.
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When pH = 1, mPEG45-b-poly(MA-iAsn-OH)53 started to
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