Synthesis and characterization of poly(amino urea urethane)-based block

copolymer and its potential application as injectable pH/temperature-sensitive

hydrogel for protein carrier
Cong Truc Huynh
1
, Quang Vinh Nguyen, Seong Woo Kang, Doo Sung Lee
*
Theranostic Macromolecules Research Center, Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea
a r t i c l e i n f o
Article history:
Received 29 May 2012
Received in revised form
10 July 2012
Accepted 14 July 2012
Available online 31 July 2012
Keywords:
pH/temperature-sensitive hydrogels
Polyurethane
Protein release
a b s t r a c t
A series of novel block copolymer comprising of poly(ethylene glycol) (PEG) and poly(amino urea
urethane) (PAUU) was simply synthesized and characterized. The block copolymers were synthesized by
a polyaddition reaction between isocyanate groups of 1,6-diisocyanato hexamethylene with secondary
amine and hydroxyl groups of 2-hydroxyethyl piperazine and hydroxyl groups at the ends of PEG in
chloroform in the presence of dibutyltin dilaurate as a catalyst and characterized by
1
H and
13
C NMR,
FTIR and gel permeation chromatography. Copolymer aqueous solutions exhibited pH/temperature-
dependent solegel phase transitions with a sol-to-gel and a gel-to-sol phase transition corresponding
to the increasing of pH and temperature, respectively, with low concentrations. The gel regions covered
the physiological condition and could be modulated by changing PAUU fraction, molecular weight of PEG
and copolymer concentration. The copolymer hydrogel did not show cytotoxicity. After injecting the
copolymer solution subcutaneously into SD rats, an in situ gel formed rapidly. The copolymer hydrogel
was conrmed as a protein depot carrier; it showed a sustained release of FITC-BSA over 6 weeks. This
novel pH/temperature-sensitive hydrogel system is a potential applicable candidate for protein carrier.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Stimuli-sensitive injectable polymeric hydrogels have potential
biomedical and pharmaceutical applications in drug and protein
delivery or tissue engineering [1e15]. Three main types of hydro-
gels have been studied: temperature-sensitive hydrogels, cationic
pH/temperature-sensitive hydrogels, and anionic pH/temperature-
sensitive hydrogels. Typical examples of temperature-sensitive
injectable hydrogels are block copolymers of hydrophilic poly(-
ethylene glycol) (PEG) with hydrophobic blocks such as poly(-
caprolactone) (PCL) [16e21], poly(lactide-co-glycolide) (PLGA)
[22e30], poly(caprolactone-co-lactide) (PCLA) [31e35], poly(tri-
methylene carbonate) (PTMC) [36e38] or polypeptide [39e44].
Aqueous solutions of these copolymers exhibit sol-gel-sol phase
transitions with increasing temperature. Their formation of
micelles and bridged micelles and the ordered packing of bridged
micelles have been attributed to a gelation mechanism [22].
However, their natural neutral properties, in some case, may limit
their use in the delivery of ionic proteins, peptides, or DNA.
pH/temperature-sensitive hydrogels exist as sols at low pH
(cationic hydrogels) or high pH (anionic hydrogels) and contain
abundant ionized groups that can form ionic interactions with
encapsulated oppositely charged bioactive therapeutic agents but
turn into gels under physiological conditions (37

C, pH 7.4). This
enables them to serve as ionic bioactive carrier depots [2e4].
Typical examples of cationic pH/temperature-sensitive hydrogels
are block copolymers consisting of PEG and poly(b-amino ester)
(PAE), poly(amino urethane) (PAU), poly(amido amine) (PAA), or
poly(amino ester urethane) (PAEU) and oligo(amido amide/amino
ester), oligo(amino urethane) [45e56]. Aqueous polymer solutions
can exist as sols at room temperature and mildly acidic pH
(pH w6.5), but can become gels under physiological conditions or
when injected into the body. Oligosulfamethazine (OSM)-based
pentablock copolymers are anionic pH/temperature-sensitive
hydrogels that exist as copolymer solutions at room temperature
and lightly basic pH (pH w 8.0). They can form ionic complexes
with cationic growth factors or therapeutic agents and act as gel
depots under physiological conditions [57e62]. These ionic pH/
temperature-sensitive hydrogels have shown potential applica-
bility as release agents for various anticancer drugs or proteins,
* Corresponding author. Tel.: 82 31 290 7282; fax: 82 31 299 6857.
E-mail address: dslee@skku.edu (D.S. Lee).
1
Current address: Australian Institute for Bioengineering and Nanotechnology,
The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
Contents lists available at SciVerse ScienceDirect
Polymer
j ournal homepage: www. el sevi er. com/ l ocat e/ pol ymer
0032-3861/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2012.07.031
Polymer 53 (2012) 4069e4075
such as chlorambucil [48,49], doxorubicin [51,55,56], paclitaxel
[57,58], human growth hormone [52], and insulin [46,54]. They
have also been studied with respect to tissue engineering [59].
Tertiary amine groups in cationic copolymers have been repor-
ted to form good ionic complexes with anionic proteins for sus-
tained release [46,54]. Polyurethane-based copolymers are
biocompatible, slowly degraded by enzymes, and strongly
hydrogen bonded [63]. This work reports a novel, simple copolymer
of poly(ethylene glycol) (PEG) and cationic poly(amino urea
urethane) (PAUU) ([PEG-PAUU]
x
copolymer). Its aqueous solutions
exhibited pH/temperature-dependent solegel phase transitions.
With the introduction of urea groups, the hydrophobicity of
copolymer was enhanced; therefore, the possibility to form
hydrophobic interaction with drug/protein could be improved and
the gelation could be achieved with low concentration of copol-
ymer that may decrease the possibility of systematic cytotoxicity of
the implanted biomaterials. Moreover, the huge change of the gel
regions can be achieved with the small change in the composition
of the copolymers. The copolymer was tested as a low-cytotoxic,
pH/temperature-sensitive hydrogel for the sustained release of
a model protein, FITC-BSA.
2. Materials and methods
2.1. Materials
Poly(ethylene glycol)s (PEG), 2-hydroxyethyl piperazine (HP),
1,6-diisocyanato hexamethylene (HDI), dibutyltin dilaurate (DBTL),
anhydrous chloroform, FITC-BSA and phosphate buffer saline (PBS)
were obtained fromSigmaeAldrich (St. Louis, MO, USA) and used as
received. Hydrochloric acid (HCl), sodium hydroxide (NaOH) and
diethyl ether were all products of Samchun Co. (Seoul, Korea). All
other reagents were of analytical grade and used as received.
2.2. Synthesis of [PEG-PAUU]
x
block copolymers
The [PEG-PAUU]
x
block copolymer was synthesized by a poly-
addition reaction between isocyanate groups of HDI with
secondary amine and hydroxyl groups of HP and hydroxyl groups at
the ends of PEG in chloroform in the presence of DBTL as a catalyst
(Scheme 1). The synthetic processing of PAUU-01 copolymer is as
follows: PEG (1 mmol) and 0.002 g of DBTL were dried in a 250 mL
two-neck round-bottom ask under vacuum at 100

C for 2 h. The
temperature was decreased to 50

C and HP (10 mmol) was added
and dried under vacuum for 30 min. The temperature of the reac-
tion mixture was cool down to room temperature and the vacuum
was replaced with dried nitrogen followed by the addition of 80 mL
anhydrous chloroform. After the reactants had dissolved, HDI
(11 mmol) was added and the reaction was continued at room
temperature for 30 min and then at 60

C for a further 3 h. Finally,
the reaction solution was concentrated by vacuumevaporation and
precipitated in excess of diethyl ether. The precipitated copolymer
was ltered and dried under vacuum for 48 h. The nal yield was
approximately 90%. The resulting [PEG-PAUU]
x
block copolymer
was characterized by
1
H and
13
C NMR, FTIR, and gel permeation
chromatography (GPC). The copolymer with different PAUU frac-
tion was obtained by changing the molecular weight of PEG and
reactants ratio.
2.3. Characterization
A 500 MHz spectrometer (Varian Unity Inova 500NB instru-
ment) was used to record the NMR spectra with CDCl
3
as the
solvent. An FT-IR spectrometer (FT/IR-4100 Type A, TGS, Jasco) was
used to record the infrared spectra.
The molecular weights of the copolymers and their distributions
were measured by gel permeation chromatography (GPC) using
a Waters Model 410 instrument with a refractive index detector
(Shodex, RI-101) and three Styragel (KF-803, KF-802.5 and KF-802)
columns in series, at a ow rate of 1.0 mL/min (eluent: CHCl
3
;
35

C). Poly(ethylene glycol) standards (Waters) were used for
calibration.
pH titration was carried out to determine the pH-sensitive
properties (pK
a
value) of the synthesized copolymer. Briey, the
copolymer was dissolved in 50 mL distilled water at a concentra-
tion of 1 mg/mL and the pH was adjusted to around 3.0 by adding
1N HCl. The titration prole was recorded by the repeated addition
of 50 mL of 0.1N NaOH. The pK
a
value was calculated from the
derivative of the titration curve, which corresponds to the inec-
tion point [55].
2.4. In vitro solegel phase transition measurement
The sol (ow)-gel (non-ow) phase transition of the copolymer
in aqueous solution was determined using the tube inversion
method. Briey, the copolymer was dissolved in phosphate buff-
ered saline (PBS) solution at pH 1 (by adding some HCL) in a 4 mL
vial (10 mm diameter) at a given concentration for 4 h. The pH was
adjusted to the desired value with 5N NaOH and 5N HCl and the
sample was stabilized at 2

C overnight. The vials, which contained
approximately 0.5 mL of the copolymer solution, were placed in
a temperature-controlled water-bath and heated slowly from 0

C
to 95

C. The samples were equilibrated for 10 min at temperature
intervals of 2

C. The solegel transition was determined by
inverting the vials [49e52].
2.5. Rheological measurement
A dynamic mechanical analyzer (Bohlin Rotational Rheometer)
was used to determine the variation of the viscosity of the copol-
ymer aqueous solutions. Oscillation mode with a controlled stress
of 0.4 Pa and a frequency of 1 rad/s was used. The copolymer
solution in PBS was placed between a 20 mm diameter plate and
a 100 mm diameter plate with a gap of 250 mm. The temperature
was increased slowly from 0 to 70

C at a heating rate of 2

C/min
[50e54].
2.6. In vitro cytotoxicity
The in vitro cytotoxicity of the hydrogels was examined by the
direct contact method. L929 broblast cells (obtained from Korean
cell line bank) were placed in a 96-well plate at a seeding density of
5000 cells per well in 0.2 mL of growth medium and maintained at
37

C for 24 h. The growth mediumwas removed and fresh medium

containing the desired amount of polymer was added. The cells
were incubated for 48 h, after which their metabolic activity was
measured with the MTT assay. Briey, the growth medium was
removed and 100 mL of fresh growth mediumcontaining 50 mg MTT
(SigmaeAldrich, St. Louis, MO, USA) was added to each well and the
cells were incubated at 37

C for 4 h. The absorbance at 570 nm
(SpectraMax

M5 Microplate Reader, Molecular Devices, Inc.) was

directly proportional to the number of living cells. The survival
percentage relative to the mock-treated cells (100% survival) was
calculated [64e66].
2.7. In vivo experiments
Male SpragueeDawley (SD) rats (Hanlim Experimental Animal
Laboratory, Seoul, Korea) were used for the in vivo experiments. The
rats (5e6 weeks old, average body weight 200 g) were handled in
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4070
accordance with the National Institutes of Health (NIH) guidelines
for the care and use of laboratory animals (NIH publication 85e23,
revised 1985). For checking the in vivo gelation, copolymer solution
(200 mL) was injected subcutaneously into SD rat. After 5 min, the
rat was sacriced and the gel morphology was observed [55].
2.8. In vitro release of FITC-BSA from PAUU hydrogel
FITC-BSA was added into the copolymer aqueous solution at pH
5.5 at the nal concentration of 1 mg/mL. The protein-copolymer
mixture solution was stirred at 4

C in 6 h for forming the ionic
complex between ionized tertiary amine groups in copolymer and
anionic groups in protein. Then, the pH of the solutions was
adjusted to 7.4 with small amounts of 5N NaOH, and 0.5 mL of the
FITC-BSA-loaded solutions was placed into 4 mL vials and incu-
bated at 37

C for 30 min to allowgel formation. Subsequently, 3 mL

of fresh release medium(PBS buffer solution, 37

C and pH 7.4) was
added to each vial, which was then incubated at 37

C and shaken
in a shaking water bath (20 strokes/min, BS-21, Jeiotech). At a pre-
determined time, 1.5 mL of the release medium was sampled and
1.5 mL of the fresh release medium was added to the vials to
maintain a constant volume of release medium. The FITC-BSA
ppm
1.0 2.0 3.0 4.0 5.0 6.0 7.0
N
O O N
H
H
N
O
O
N
H
N
H
O O
6
m
x
O
n
N
b c d
b c d
f e
f e
g
h
a
a
a e b c d
CDCl
3
h g f
ppm
25.0 50.0 75.0 100.0 125.0 150.0
c d
CDCl
3
a,
a
a
N
O O N
H
H
N
O
O
N
H
N
H
O O
6
m
x
O N
b c d
b c d
f e
f e
g
h
i
i
n-2
a
a
O
O
a'
a"
i
b
f e
g
h
a
b
Fig. 1. (a)
1
H and (b)
13
C NMR spectra of PAUU-01 copolymer.
OCN NCO
6
OH
N HN
HDI HP
HO
O
H
n
+
PEG
DBTL
CHCl3
[PEG-PAUU]
x
N
O O N
H
H
N
O
O
N
H
O
O
n
N
x
H
N
O
m
+
Scheme 1. Synthesis route of [PEG-PAUU]
x
block copolymer.
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4071
concentration in the release media and standard samples was
analyzed by UVeVis spectroscopy (SpectraMax

M5 Microplate
Reader, Molecular Devices, Inc.) at 494 nm [67]. The absorbance
comparison was used to calculate the FITC-BSA concentration and
accumulative release with the known FITC-BSA concentrations
ranging from 100 to 0.01 mg/mL.
2.9. Circular dichroism (CD) spectroscopy
To conrm the stability of FITC-BSA in in vitro experiment, CD
measurement was carried out. CD spectra were obtained on Jasco J-
810(Jasco, Tokyo, Japan) equippedwithawater bathoperatedat room
temperature (25

C). A quartz cuvette with 1 mm path length was
used and the spectra were scanned between 200 and 260 nm [54].
3. Results and discussion
3.1. Synthesis and characterization of [PEG-PAUU]
x
block copolymer
The [PEG-PAUU]
x
block copolymer was synthesized by a poly-
addition reaction between isocyanate groups of HDI with
secondary amine and hydroxyl groups of HP and hydroxyl groups at
the ends of PEG in chloroform in the presence of DBTL as a catalyst
(Scheme 1). Fig. 1 shows the
1
H and
13
C NMR spectra with labeled
protons and carbons of PAUU-01 copolymer. As shown in Fig. 1a, the
protons at 3.51e3.74 ppm (peak a) were assigned to the methylene
proton of PEG. The protons at 3.02e3.22 ppm (peak c) and those at
1.25e1.58 ppm (peaks d and e) were assigned to the rst, second
and third methylene protons of HDI, respectively. The presence of
peak e, f, g and h demonstrated the presence of HP in the copol-
ymer. The fraction of PAUU was calculated by the relative peak
areas of peak a (PEG) and peak f (HP in PAUU). Fig. 1b shows the
13
C
NMR spectrum of the PAUU-01 copolymer with labeled carbons.
The isocyanate peak at 122.9 ppmwas not observed, indicating that
the HDI monomer had been consumed completely during
polymerization.
The formation of the copolymers structure was also conrmed
further by FTIR spectroscopy (Fig. 2). As shown in Fig. 2a, the peak
at 3334 cm
1
(#1) corresponded to the eNHe stretching vibration
of urea and urethane groups. HDI had been consumed completely
because of the absence of a peak at 2267 cm
1
(#2). The peaks at
1675 and 1725 cm
1
(#3) showed the stretching of the urea and
urethane groups and the CeOeC ether stretching of PEG showed
peaks at 1104 cm
1
(#4). Fig. 2b shows the peaks for the carbonyl
groups with and without hydrogen bonding (peak #3 in Fig. 2a),
further demonstrating the formation of functional urethane groups.
Furthermore, the molecular weight of the copolymers and their
distributions were determined by GPC using chloroform as the
solvent and PEG for calibration. The GPC curves of the synthesized
copolymers show a unimodal peak with relative low values of
molecular weight distributions. The above characterizations clearly
demonstrate the successful synthesis of the [PEG-PAUU]
x
block
copolymers. In addition, the pH-sensitive properties (pK
a
) of the
synthesized copolymer were determined by the pH titration
method. The pK
a
[PEG-PAUU]
x
block copolymers are relative low
(5.65e5.89). Table 1 lists the detailed characteristics of the
synthesized copolymers.
Fig. 3. Sol-gel phase transition diagram of PAUU-01 copolymer hydrogel.
Fig. 2. (a) FTIR spectrum of copolymer PAUU-01 (eNHe stretching vibration of urea and urethane groups: #1, 3334 cm
1
; disappearance of the eNCO groups stretching vibration:
#2, 2267 cm
1
; stretching vibration of the carbonyl of urea and urethane groups: #3, 1720 cm
1
; ether groups of PEG stretching: #4, 1104 cm
1
) and (b) stretching vibration of the
functional carbonyl of urea and urethane groups.
Table 1
Characteristics of the synthesized [PEG-PAUU]
x
block copolymer.
Sample M
n
a
(PEG)
Feed ratio
PEG/HP/HDI
[PEG-PAUU
b
]
x
c
Fraction
PAUU
b
(wt%)
M
n
c
PDI
c
pK
a
d
PAUU-01 4600 1/10/11 [4600e2400]
2.79
34.3 19500 1.61 5.65
PAUU-02 4600 1/7/8 [4600e1660]
3.18
31.6 19900 1.84 5.89
PAUU-03 2000 1/4/5 [2000e1050]
6.95
34.4 21200 2.08 5.87
a
Provided by Sigma.
b
Calculated by
1
H NMR.
c
Measured and calculated by GPC.
d
Calculated from titration curves.
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4072
3.2. Sol-gel phase transition
The pH- and temperature-dependence of the solegel phase
transition were measured by tube inverting [49e55]. Fig. 3 shows
the solegel phase transition diagram of PAUU-01 hydrogels with
the existence of the gel (>7.5 wt%) at physiological conditions
(37

C, pH 7.4). By introduction the urea groups, the gelation could
be achieved with low concentration of copolymer that may
decrease the possibility of systematic cytotoxicity of the implanted
biomaterials. The [PEG-PAUU]
x
block copolymer showed a similar
sol-to-gel phase transition to PAE-PEG-PAE triblock copolymers and
[PEG-PAEU]
x
block copolymers with increasing temperature. Its
transition was different from that of [PEG-PAU]
x
multiblock copol-
ymer, which showed a sol-to-gel-to-sol phase transition [47e49].
The difference was attributed to the presence of strongly hydrogen
bonding urea groups in the PAUU-based copolymer. The aqueous
PAUU-01 copolymer was a sol at every tested temperature at lowpH
(e.g. pH 5.5) because of the ionization of the tertiary amine groups,
resulting in the PAUU segments and copolymer being hydrophilic
[47e52]. At the basic pH (e.g. pH 7.4, 15 wt%), the copolymer solu-
tionwas a gel at lowtemperature because the tertiary amine groups
of the PAUU segments were completely deionized (pK
a
< 6.0),
allowing strong hydrophobic interactions and hydrogen bonds to
form between the deionized PAUU segments [47e52]. At the basic
pH, increasing temperature induced a gel-to-sol transition due to
the breaking of networks caused by the breaking of hydrogen bonds
between urea and urethane groups in the PAUU segments and the
partial dehydration of PEG at high temperatures [47,50e52].
Fig. 4 shows photographs of the in vitro solegel phase transition
of 15 wt% PAUU-01 copolymer hydrogel with changing pH and
temperature. At lowpH and roomtemperatures (e.g. 20

C, pH 5.5),
the tertiary amine groups in copolymer were ionized result in the
hydrophilic copolymer that lead to the existence of a solution, as
shown in Fig. 4a. However, at the physiological conditions (37

C,
pH 7.4), the hydrophobicity of copolymer caused by de-ionized
tertiary amine groups and strong hydrogen bond between urea
and urethane groups led to the formation of the gel, as shown in
Fig. 4b. This solegel transition is a reversible process.
The gel region of [PEG-PAUU]
x
copolymer hydrogel can be
controlled through copolymer composition and its aqueous
concentration (Figs. 3 and 5). As shown in Fig. 3, lowering copol-
ymer concentrations can shift the gel region to lower temperature
and higher pH due to the decrease of hydrogen bonds and hydro-
phobic interactions density [52]. Fig. 5 shows the inuence of PAUU
fraction (or PAUU block length) (PAUU-01 and PAUU-02) and
molecular weight of PEG (PAUU-01 and PAUU-03) on the solegel
phase transition. At the same molecular weight of PEG at 4600,
with decreasing PAUU block length from 2400 to 1660 (PAUU-01 to
PAUU-02), the huge change in the gel region was observed that it
shifted to much lower temperature and higher pH (Fig. 5a) or
concentration (Fig. 5b) because of the decrease in density of func-
tional groups in solution. On the other hand, at the same PAUU
fraction of 34.4%, the gel region shifted to higher temperature and
lower pH (Fig. 5a) or lower concentration (Fig. 5b) with increasing
molecular weight of PEGfrom2000 to 4600 (PAUU-03 to PAUU-01).
It was attributed to the larger size of micelles [51e53].
3.3. Rheological properties
Dynamic rheological analyzer was used to examine the variation
in the mechanical properties of the synthesized [PEG-PAUU]
x
copolymer hydrogels. Fig. 6 shows the change in viscosity of 15 wt%
PAUU-01 solutions as a function of temperature at different pH. The
viscosity of the copolymer solution at pH 5.5 increased with
increasingtemperaturethoughnot enoughtomakethe sol tobea gel.
However, at pH7.4, theviscosity(w10
3
Pa s) indicates theexistenceof
the gel state, which was conrmed by the tube-inverting method
[51e53]. Similar trends were observed at pH 6.5 and 7.0. A guide for
the eye about the change of the copolymer solution viscosity upon
injectionintothe bodywas depictedby the (red) dashed-lineinFig. 6.
The injectionof low-viscositycopolymer solutionat pH5.5 and room
temperature (fromA, w1 Pa s) resultedina highviscositygel forming
under physiological conditions (to B, w10
3
Pa s).
3.4. In vitro cytotoxicity
The in vitro cytotoxicity of the PAUU-based copolymer was
examined by the direct contact method using L929 broblast cells
Fig. 5. Sol-gel phase transitions of PAUU-based hydrogel with the inuence of PAUU block length (PAUU-01 and PAUU-02) and molecular weight of PEG (PAUU-01 and PAUU-03):
(a) copolymer 15 wt% at different pH and (b) different copolymer concentration at pH 7.4.
Fig. 4. In vitro solegel phase transition of 15 wt% PAUU-01 copolymer hydrogel with
changing pH and temperature: (a) solution state (20

C, pH 5.5) and (b) gel state (37

C,
pH 7.4).
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4073
and MTT assay. The [PEG-PAUU]
x
copolymer was shown to be low-
cytotoxic at high concentrations of copolymer. As shown in Fig. 7,
cell viabilitywas 90%andw70%at copolymer concentrationof 1000
and 2000 mg/mL, respectively, indicating that [PEG-PAUU]
x
block
copolymer could be used as a low-cytotoxic biomaterial [65,66].
3.5. In vivo gel formation
The injectability and in vivo gelation of [PEG-PAUU]
x
hydrogel
were examined. 200 mL 15 wt% copolymer solution at pH 5.5 was
subcutaneously injected into an SD rat using a syringe needle
(26G). After 5 min, the rat was sacriced and a strong white gel was
observed. As shown in Fig. 8, the gel formed quickly due to the
change of pH and temperature under physiological conditions. This
suggests that the [PEG-PAUU]
x
copolymer hydrogel could be used
as an injectable hydrogel system with a short gelation time.
3.6. In vitro release of FITC-BSA and stability of the released BSA
The potential applicability of [PEG-PAUU]
x
hydrogel as a protein
carrier for long-term sustained release was tested in vitro via the
release of FITC-BSA. Fig. 9 shows the sustained release of FITC-BSA
from the [PEG-PAUU]
x
hydrogel for over 6 weeks. It shows that the
release mechanism is diffusion controlled at the rst 10 days and
then the combination of diffusion and surface erosion. The sus-
tained release of protein was attributed to the formation of ionic
complexes and strong hydrogen bonds between the cationic poly-
mer and the anionic protein [46,52]. In addition, the stability of the
released FITC-BSA was conrmed by CD measurement. After 2 and
6 weeks, the released FITC-BSA retained its secondary structure
Time (day)
0 10 20 30 40
C
u
m
u
l
a
t
i
v
e

r
e
l
e
a
s
e

o
f

F
I
T
C
-
B
S
A

(
%
)
0
20
40
60
80
100 Gel 10 wt%
Gel 15 wt%
Fig. 9. In vitro sustained release of FITC-BSA from PAUU-01 hydrogel.
Fig. 8. (a) In vivo gel morphology 5 min after subcutaneous injection of 200 mL 15 wt%
PAUU-01 copolymer solution (at pH 5.5) into an SD rat. (b) The gel after tissue removal.
Copolymer concentration (g/mL)
Control 50 100 500 1000 2000
C
e
l
l

v
i
a
b
i
l
i
t
y

(
%
)
0
20
40
60
80
100
120
Fig. 7. In vitro cytotoxicity of PAUU-01 copolymer at different copolymer concentration
using L929 broblast cells.
Temperature (
o
C)
0 10 20 30 40 50 60
V
i
s
c
o
s
i
t
y

(
P
a

s
)
10
-1
10
0
10
1
10
2
10
3
10
4
pH 7.4
pH 7.0
pH 6.5
pH 5.5
(Injected point) A
(Body condition) B
Fig. 6. Variation in viscosity of 15 wt% PAUU-01 copolymer with respect to tempera-
ture at different pH.
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4074
(the band at 209 nm primarily corresponds to the alpha helical
structure whereas the band at 222 nm is assigned mainly to the
beta structure), as shown in Fig. 10, indicating that the formation of
ionic complex between protein and copolymer hydrogel may
protect protein from denaturation or the conformational changes
caused by the shielding effect for long-term sustained release [54].
These results conrm the potential applicability of [PEG-PAUU]
x
hydrogel as a protein carrier for long-term sustained release.
4. Conclusion
A novel, simple structure copolymer based on poly(amino urea
urethane) block copolymer was prepared and used as injectable pH/
temperature-sensitive hydrogel system. Its aqueous solution was
a sol at roomtemperatureandmildlyacidic pH(20

C, pH5.5), which
is suitable for mixingwithproteins or bioactive molecules. However,
its aqueous solution became a gel under physiological conditions
(37

C, pH7.4) or after being injectedintothe body, whichcouldlet it

serve as a protein depot for long-termrelease. With the presence of
the urea group, the aqueous copolymer solution can gel at low
copolymer concentrationanda hugechange of thegel regions canbe
achieved with the small change in the composition of the copoly-
mers. This [PEG-PAUU]
x
hydrogel shows strong mechanical prop-
erties, long-term stability in vitro (more than 6 weeks) and non-
cytotoxicity that may allow its application as a biomaterial.
Specially, the sustained release of FITC-BSA from [PEG-PAUU]
x
hydrogel in vitro for up to 6 weeks highlights its possible applica-
bility for the long-term sustained release of proteins.
Acknowledgments
This study was supported by the Basic Science Research Program
through a National Research Foundation of Korea (NRF) grant
funded by the Korean Government (MEST) (2010-0027955) and the
Converging Research Center Program funded by the Ministry of
Education, Science and Technology (2011K000817).
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Wavelength (nm)
200 210 220 230 240 250
C
D

(
m
d
e
g
)
-20
-10
0
10
20
Released FITC-BSA from 15% gel (2 weeks)
Released FITC-BSA from 15% gel (6 weeks)
Original FITC-BSA (0.1 mg/mL)
Fig. 10. CD spectra of original and released FITC-BSA.
C.T. Huynh et al. / Polymer 53 (2012) 4069e4075 4075