Dually cationic and anionic pH/temperature-sensitive injectable hydrogels and potential application as a protein carrier

Article abstract of DOI:10.1039/c2cc36134e

Novel copolymers containing both anionic and cationic pH-sensitive moieties were reported. These amphoteric copolymers exhibited special closed-loop reversible sol-gel-sol phase transitions in response to both pH and temperature.

Full text of DOI:10.1039/c2cc36134e

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Chemical Communications
www.rsc.org/chemcomm
Volume 48 | Number 89 | 18 November 2012 | Pages 10921–11036
ISSN 1359-7345
COMMUNICATION
Doo Sung Lee et al.
Dually cationic and anionic pH/temperature-sensitive injectable hydrogels and potential application
as a protein carrier

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COMMUNICATION
Dually cationic and anionic pH/temperature-sensitive injectable
hydrogels and potential application as a protein carrierw
Cong Truc Huynh,z Minh Khanh Nguyeny and Doo Sung Lee*
Received 23rd August 2012, Accepted 23rd September 2012
DOI: 10.1039/c2cc36134e
Novel copolymers containing both anionic and cationic pH-sensitive
moieties were reported. These amphoteric copolymers exhibited
special closed-loop reversible sol–gel–sol phase transitions in
response to both pH and temperature.
pH/temperature-responsive therapeutic delivery provides a promis-
ing strategy for on demand release of a variety of bioactive factors.
Injectable pH/temperature-responsive hydrogel systems capable of
forming an ionic complex with oppositely charged bioactive factors
have been widely explored for bioactive agent delivery vehicles.1–3
Among them, anionic hydrogels may offer potential for complexa-
tion with cationic molecules (e.g., bone morphogenetic protein
(BMP), transforming growth factor beta (TGF-b), etc.),4 whereas
cationic hydrogels may interact with anionic macromolecules (e.g.,
insulin, human growth hormone (hGH), etc.).5–8 The ionic inter-
action may retain the therapeutics in the hydrogels. pH-responsive
polymeric hydrogels based on poly(2-(diisopropylamino) ethyl
methacrylate) (PDPAEMA), poly(2-(diethylamino) ethyl methacry-
late) (PDEAEMA), poly(2-(dimethylamino) ethyl methacrylate)
(PDMAEMA), poly(N-isopropylacrylamide-co-propylacrylic acid)
(p(NIPAAm-co-PAAc)) copolymers, poly(NIPAAm-co-PAAc-co-
butyl acrylate) (p(NIPAAm-co-PAAc-co-BA)), poly(amidoamine)
(PAA), poly(amino urethane) (PAU) and poly(amino urea
urethane) (PAUU) have been reported.9,10 These materials display
a non-biodegradability that may limit their practical applications.
To overcome this shortcoming, different series of pH/temperature-
responsive polymers based on oligosulfamethazine (OSM), poly-
(b-amino ester) (PAE) and poly(b-amino ester urethane) (PAEU)
have been developed as biodegradable hydrogel systems.6–8,11–13
Ionic biomolecules are retained within these pH/temperature-
responsive hydrogels that will hydrolytically degrade to release
the payloads. However, these hydrogels are either cationic or
Scheme 1 Chemical structure of PUASM copolymer.
anionic, because the materials only bear basic (amine) or
acidic (sulfonamide) groups.
Here, we introduce an innovative class of amphoteric
copolymers, poly(urethane amino sulfamethazine)-based block
copolymers (PUASM), capable of forming dually cationic and
anionic hydrogels in response to pH and temperature. The
copolymer aqueous solutions exhibited as a free flowing sol
at both mildly acidic and basic pH, but a gel was induced at a
pH range of 6.8–8.2 as temperature increased. The dually
cationic and anionic characteristics, mechanical properties,
cytotoxicity and potential application as a protein carrier of
the amphoteric hydrogels were examined.
The PUASM block copolymers (Scheme 1) were synthesized
by polyaddition of isocyanate groups of 1,6-diisocyanato
hexamethylene (HDI) with hydroxyl groups of a synthesized
dihydroxyl amino sulfamethazine monomer (DHASM) and of
triblock poly(e-caprolactone-lactide)-poly(ethylene glycol)-poly-
(e-caprolactone-lactide) (PCLA-PEG-PCLA) copolymers in the
presence of dibutyltin dilaurate (DBTL) as a catalyst (Scheme S1
in ESIw).12 The PCLA-PEG-PCLA triblock copolymers were
synthesized and characterized as in the previous report.11 The
DHASM was prepared by Michael-addition reaction12 between
the secondary amine groups of diethanolamine (DEA) and the
vinyl groups of acrylated sulfamethazine (SM-A), which was
similarly synthesized to the previous procedure.11 The synthesized
1
PUASM block copolymers were characterized using H NMR
(Fig. S1 in ESIw) and gel permeation chromatography (GPC). The
detailed characteristics of the synthesized PUASM copolymers
are listed in Table S1 in ESI.w
Theranostic Macromolecules Research Center, Department of
Polymer Science and Engineering, Sungkyunkwan University, Suwon,
Gyeonggi-do 440-746, South Korea. E-mail: dslee@skku.edu;
Fax: +82 31 299 6857; Tel: +82 31 290 7282
w Electronic supplementary information (ESI) available: Detailed
experimental procedures, characterization, sol–gel transition, degrada-
tion and cell cytotoxicity. See DOI: 10.1039/c2cc36134e
z Present address: Australian Institute for Bioengineering and Nano-
technology, The University of Queensland, St Lucia, Brisbane, QLD
4072, Australia.
To confirm the dual ionic properties of the synthesized ampho-
teric copolymer, zeta potentials of the synthesized amphoteric
PUASM, cationic PAE-based (PAE-poly(e-caprolactone)-PEG-
poly(e-caprolactone)-PAE, PAE-PCL-PEG-PCL-PAE:1250-1500-
1650-1500-1250)6 and anionic OSM-based (OSM-PCLA-PEG-
PCLA-OSM:1140-1550-1750-1550-1140)4 copolymer solutions
were compared. As shown in Fig. 1, zeta potential of the cationic
PAE-based copolymer solutions decreased with increasing pH.
y Present address: Department of Biomedical Engineering, Case Western
Reserve University, Cleveland, OH 44106, USA.
c
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Chem. Commun., 2012, 48, 10951–10953 10951

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Fig. 3 Schematic showing reversible sol–gel–sol phase transition of
PUASM hydrogels with changing temperature and pH. Ionization of basic
portions containing amine groups in the PASM at slightly acidic pH (a) and
of acidic portions composed of sulfonamide groups in the PASM at slightly
basic pH (b) renders the polymer hydrophilic, resulting in a sol state in
water. In contrast, relative deionization of both basic and acidic
portions of the PASM and increases in hydrophobicity of the PCLA
under physiological conditions led to formation and interconnection of
nanostructured polymeric micelles, resulting in gelation (c). The positive
and negative charges in c indicate that the PASM is not fully deionized.
Fig. 1 Zeta potential of amphoteric PUASM (PUASM-2), cationic
PAE-based (PAE-PCL-PEG-PCL-PAE) and anionic OSM-based
(OSM-PCLA-PEG-PCLA-OSM) copolymers in water (2 mg mLꢀ1
)
at different pH.
It was +19.5 mV at pH 6.5, +2.7 mV at pH 7.5, and +1.0 mV
at pH 8.0. In contrast, zeta potential of the anionic OSM-based
copolymer solutions increased when lowering pH. Interestingly,
as a combination, zeta potential of the amphoteric PUASM
copolymer solutions was positive at acidic pH (+4.6 mV at
pH 6.0), and became negative at basic pH (ꢀ11.4 mV at pH 8.0).
This is a result of ionization of the tertiary amine at relatively
acidic pH and sulfonamide groups at relatively basic pH, and
their deionization at neutral pH.
completely clear. In this amphoteric PUASM hydrogel system,
the combination of anionic and cationic pH-sensitive moieties
accounted for the closed-loop sol–gel–sol phase transition
mechanism that is depicted in Fig. 3. Specifically, the ionization
of tertiary amine groups at relatively acidic pH (e.g., pH 6.5)
or sulfonamide groups at relatively basic pH (e.g., pH 8.5)
rendered the copolymers hydrophilic, resulting in sol states in
the temperature range of 0–65 1C. At a pH range of 6.8–8.2,
even though the PASM domains (including both cationic and
anionic portions) became more hydrophobic as a result of an
increase in degree of their deionization, the sol state still existed
at low temperatures, due to the less hydrophobicity of the
PCLA segments.7,11 In this stage, the hydrophobic interactions
among the PASM were not sufficient to induce gelation. In
contrast, an increase in the hydrophobicity of the PCLA at
higher temperatures led to formation of micelles bridged by
PEG segments, and thus gelation occurred. The gel-to-sol
transition appeared with further increasing temperature, which
is relevant to the partial dehydration of PEG segments and the
breakage of hydrophobic interactions.11–13 The closed-loop gel
regions of the PUASM hydrogels are adjusted by varying the
molecular weight of the PEG block or copolymer concentration
(Fig. S2 in ESIw).11–13,17
The PUASM copolymers displayed special closed-loop
reversible sol–gel–sol phase transition, determined using the
tube inversion method,13 when varying pH and temperature,
as shown in Fig. 2. This behaviour results from the amphoteric
properties of the copolymers combining anionic sulfonamide
and cationic tertiary amine groups in the poly(amino sulfa-
methazine) (PASM) blocks. The closed-loop sol–gel phase
transition has been reported in previous literature.14–16 Thermo-
sensitive copolymers of PEG and poly(ethyl-2-cyanoacrylate)
(PEC) exhibited a closed-loop sol–gel–sol phase transition as
a function of temperature and concentration.14 Multiblock
copolymers composed of a poloxamer (P88) and terephthalic
anhydride showed a closed-loop sol–gel–sol phase transition
at below physiological conditions relevant to the anionic
properties of the copolymer.15 Recently, the similar closed-
loop sol–gel–sol phase transition was observed with Pluronic
F127 end-capped by the polyamine hydrogel system.16 However,
the mechanism of their gel-to-sol transition at high pH was not
Variation in viscoelasticity of the PUASM copolymer aqueous
solutions at different pH values as a function of temperature was
examined using dynamic rheological analysis.12 As shown in
Fig. 4, while the copolymer solutions exhibited low viscosities
Fig. 2 Closed-loop sol–gel phase diagram of PUASM-2 hydrogel
(25 wt%). The bioactive molecules and copolymer solution can be
formulated and injected into the body at either slightly acidic pH (e.g., A,
pH 6.8) or slightly basic pH (e.g., B, pH 8.0), and a gel forms rapidly
(see Fig. S3 in ESIw) under physiological conditions (C, 37 1C, pH 7.4).
Fig. 4 Variation in viscosity of 25 wt% PUASM-2 copolymer solu-
tions at different pH as a function of temperature.
c
10952 Chem. Commun., 2012, 48, 10951–10953
This journal is The Royal Society of Chemistry 2012

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may provide a promising versatile approach for bioactive
molecules delivery.
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 Bio & Medical Technology Develop-
ment Program of the National Research Foundation (NRF)
funded by the Korean Government (MEST) (2011-0019391).
Notes and references
1 (a) C. T. Huynh, M. K. Nguyen and D. S. Lee, Macromolecules, 2011,
44, 6629; (b) M. K. Nguyen and D. S. Lee, Macromol. Biosci., 2010,
10, 563; (c) L. Yu and J. Ding, Chem. Soc. Rev., 2008, 37, 1473.
2 (a) B. Jeong, Y. H. Bae, D. S. Lee and S. W. Kim, Nature, 1997,
388, 860; (b) M. H. Park, M. K. Joo, B. G. Choi and B. Jeong, Acc.
Chem. Res., 2012, 45, 424; (c) U. P. Shinde, M. K. Joo, H. J. Moon
and B. Jeong, J. Mater. Chem., 2012, 22, 6072; (d) G. Chang,
L. Yu, Z. Yang and J. Ding, Polymer, 2009, 50, 6111.
3 (a) A. S. Hoffman, Adv. Drug Delivery Rev., 2002, 53, 3; (b) Z. M.
O. Rzaev, S. Dinc¸ er and E. Pi-skin, Prog. Polym. Sci., 2007, 32, 534.
4 H. K. Kim, W. S. Shim, S. E. Kim, K.-H. Lee, E. Kang, J.-H. Kim,
K. Kim, I. C. Kwon and D. S. Lee, Tissue Eng., Part A, 2009,
15, 923.
5 M. K. Nguyen, C. T. Huynh, G. H. Gao, J. H. Kim, D. P. Huynh,
S. Y. Chae, K. C. Lee and D. S. Lee, Soft Matter, 2011, 7, 2994.
6 D. P. Huynh, M. K. Nguyen, B. S. Pi, M. S. Kim, S. Y. Chae,
K. C. Lee, B. S. Kim, S. W. Kim and D. S. Lee, Biomaterials, 2008,
29, 2527.
7 D. P. Huynh, G. J. Im, S. Y. Chae, K. C. Lee and D. S. Lee,
J. Controlled Release, 2009, 137, 20.
8 C. T. Huynh, S. W. Kang, Y. Li, B. S. Kim and D. S. Lee, Soft
Matter, 2011, 7, 8984.
9 (a) Y. Ma, Y. Tang, N. C. Billingham and S. P. Armes, Biomacro-
molecules, 2003, 4, 864; (b) M. D. Determan, J. P. Cox and
S. K. Mallapragada, J. Biomed. Mater. Res., Part A, 2007, 81, 326;
(c) F. Bossard, T. Aubry, G. Gotzamanis and C. Tsitsilianis, Soft
Matter, 2006, 2, 510; (d) J. C. Garbern, A. S. Hoffman and
P. S. Stayton, Biomacromolecules, 2010, 11, 1833; (e) J. C. Garbern,
E. Minami, P. S. Stayton and C. E. Murry, Biomaterials, 2011, 32, 2407.
10 (a) C. T. Huynh, Q. V. Nguyen, S. W. Kang and D. S. Lee,
Polymer, 2012, 53, 4069; (b) M. K. Nguyen, D. K. Park and
D. S. Lee, Biomacromolecules, 2009, 10, 728; (c) M. K. Nguyen and
D. S. Lee, Chem. Commun., 2010, 46, 3583; (d) C. T. Huynh,
M. K. Nguyen, D. P. Huynh, S. W. Kim and D. S. Lee, Polymer,
2010, 51, 3843.
11 (a) W. S. Shim, J.-H. Kim, H. Park, K. Kim, I. C. Kwon and
D. S. Lee, Biomaterials, 2006, 27, 5178; (b) W. S. Shim, S. W. Kim
and D. S. Lee, Biomacromolecules, 2006, 7, 1935.
12 C. T. Huynh, M. K. Nguyen, J. H. Kim, S. W. Kang, B. S. Kim
and D. S. Lee, Soft Matter, 2011, 7, 4974.
13 (a) C. T. Huynh and D. S. Lee, Colloid Polym. Sci., 2012,
290, 1077; (b) C. T. Huynh, M. K. Nguyen, D. P. Huynh and
D. S. Lee, Colloid Polym. Sci., 2011, 289, 301.
14 B. G. Choi, Y. S. Sohn and B. Jeong, J. Phys. Chem. B, 2007,
111, 7715.
Fig. 5 Concentration of hGH in the plasma of SD rats after injecting
200 mL of the hGH solutions (10 mg mLꢀ1) and 200 mL of the hGH-loaded
PUASM-2 (25 wt%) solutions (hGH 10 mg mLꢀ1) (ꢁSD, n = 5).
(o1 Pa s) at low temperatures (o20 1C) in the pH range from
6.5 to 8.5, the viscosities significantly increased with increasing
temperature to 65 1C. At pH 7.4, the viscosity markedly
increased from 0.24 Pa s (a sol state) to 2381 Pa s (a gel state)
as temperature was increased from 15 to 37 1C due to the
enhancement in hydrophobicity of PCLA. At body tempera-
ture (37 1C), the viscosities of the copolymer solutions were
2.68, 450 and 2381 Pa s corresponding to pH values at 6.5 to
7.0 and 7.4. However, they decreased to 405 and to 1.51 Pa s
with further increasing pH to 8.0 and 8.5, respectively. The low
viscosities at both acidic (pH 6.5) and basic pH (pH 8.5) may
facilitate homogeneous encapsulation of bioactive agents as
well as subcutaneous injection of the copolymer solutions into
the body (Fig. S3 in ESIw shows the photographs of the gels
at five minutes and one week after injection of PUASM
aqueous solutions at pH 6.8 and pH 8.0 into Sprague–Dawley
(SD) rats).
The possibility of releasing anionic protein from the ampho-
teric PUASM hydrogel was examined using hGH as a
model protein. An in vivo release profile of hGH from the
PUASM hydrogels in SD rats is presented in Fig. 5. The hGH
concentration in the serum of the SD rats with hGH-loaded
hydrogels was maintained at higher regarded effective concen-
tration ( Z1 ng mLꢀ1 18
for more than 3 days with a minimal
)
initial burst. Meanwhile, the hGH solution group (as a negative
control group) showed a significantly initial burst release profile.
The controlled release of hGH from the complex hydrogel was
governed by ionic complexation between the anionic hGH and
cationic moieties in the PUASM copolymer. In addition, the
PUASM hydrogel was confirmed to be a biodegradable (Fig. S4
in ESIw) and low-cytotoxic, according to the ISO/EN 10993
Part 5 Guidelines19 (Fig. S5 in ESIw), hydrogel system.
In summary, dually anionic and cationic copolymers have
been successfully synthesized. Aqueous copolymer solutions
exhibited special closed-loop reversible sol–gel–sol phase transi-
tions as a function of temperature and pH with controllable gel
regions. The aqueous copolymer solutions were administered
into SD rats at both acidic and basic pH. The biodegradability,
low-cytotoxicity, injectability and potential application as a
protein carrier of the hydrogel system suggest that the material
15 J. M. Suh, S. J. Bae and B. Jeong, Adv. Mater., 2005, 17, 118.
16 S. Y. Lee, Y. Lee, J. E. Kim, T. G. Park and C.-H. Ahn, J. Mater.
Chem., 2009, 19, 8198.
17 (a) C.-Y. Gong, S. Shi, L. Wu, M.-L. Gou, Q.-Q. Yin, Q.-F. Guo,
P.-W. Dong, F. Zhang, F. Luo, X. Zhao, Y.-Q. Wei and
Z.-Y. Qian, Acta Biomater., 2009, 5, 3358; (b) M. S. Shim,
H. T. Lee, W. S. Shim, I. Park, H. Lee, T. Chang, S. W. Kim
and D. S. Lee, J. Biomed. Mater. Res., 2002, 61, 188.
18 M.-R. Park, C.-J. Chun, S.-W. Ahn, M.-H. Ki, C.-S. Cho and
S.-C. Song, Biomaterials, 2010, 31, 1349.
19 D. Park, W. Wu and Y. Wang, Biomaterials, 2011, 32, 7.
c
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Chem. Commun., 2012, 48, 10951–10953 10953