PH-sensitive poly(histidine methacrylamide)

Article abstract of DOI:10.1021/acs.langmuir.6b01465

This research reports a synthetic amino acid based zwitterionic poly(histidine methacrylamide) (PHisMA), which possesses switchability among zwitterionic, anionic, and cationic states, pH-dependent antifouling properties, and chelation capability to multivalent metal ions. The PHisMA polymer brush surface shows good antifouling properties to resist protein adsorption and bacterial attachment in its zwitterionic state at pH 5. This study also demonstrates that the solution acidity significantly affects the mechanical properties of PHisMA hydrogels. PHisMA hydrogels show higher viscoelastic properties and lower swelling ratios in the zwitterionic state at pH 4 and pH 5, compared to higher or lower pH conditions. It was discovered that PHisMA can chelate multivalent metal ions, such as Ca²⁺, Mg²⁺, Cu²⁺, Ni²⁺ and Fe³⁺. This study provides us a better understanding of structure-property relationships of switchable zwitterionic polymers. PHisMA can potentially be adapted for a broad range of applications including wound care, water treatment, bioseparation, coating, drug and gene delivery carriers, etc.

Full text of DOI:10.1021/acs.langmuir.6b01465

Article
pubs.acs.org/Langmuir
pH-Sensitive Poly(histidine methacrylamide)
Huifeng Wang,† Haiyan Wu,† Chen-Jung Lee,† Xia Lei,† Jiang Zhe,‡ Fujian Xu,§,∥ Fang Cheng,⊥
and Gang Cheng*,†
‡
†Department of Chemical and Biomolecular Engineering and Department of Mechanical Engineering, University of Akron, Akron,
Ohio 44325, United States
§Key Laboratory of Carbon Fiber and Functional Polymers, Beijing University of Chemical Technology, Ministry of Education,
Beijing 10029, China
∥Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China
⊥School of Pharmaceutical Engineering, Dalian University of Technology, Dalian, Liaoning Province, 116024, China
ABSTRACT: This research reports a synthetic amino acid based
zwitterionic poly(histidine methacrylamide) (PHisMA), which pos-
sesses switchability among zwitterionic, anionic, and cationic states,
pH-dependent antifouling properties, and chelation capability to
multivalent metal ions. The PHisMA polymer brush surface shows
good antifouling properties to resist protein adsorption and bacterial
attachment in its zwitterionic state at pH 5. This study also
demonstrates that the solution acidity significantly affects the
mechanical properties of PHisMA hydrogels. PHisMA hydrogels
show higher viscoelastic properties and lower swelling ratios in the
zwitterionic state at pH 4 and pH 5, compared to higher or lower pH
conditions. It was discovered that PHisMA can chelate multivalent
metal ions, such as Ca2+, Mg2+, Cu2+, Ni2+ and Fe3+. This study
provides us a better understanding of structure−property relationships of switchable zwitterionic polymers. PHisMA can
potentially be adapted for a broad range of applications including wound care, water treatment, bioseparation, coating, drug and
gene delivery carriers, etc.
existing amino acid based zwitterionic materials, primary/
secondary amines respond to pH changes at basic conditions
(pH 9−12). In many applications, such as wound healing, gene
delivery and antimicrobial applications, materials that can
respond to pH at neutral and slightly acidic conditions are
needed. In our previous study, we developed two zwitterionic
carboxybetaine polymers carrying tertiary amines as the
cation,39 which have buffering capacities at pH 7−9.
The object of this work is to develop a pH-responsive
zwitterionic poly(histidine methacrylamide) (PHisMA) that
can respond to pH at acidic (5−6) conditions and understand
the structure−property relationships of PHisMA under differ-
ent pH conditions. Due to the unique properties of imidazole
group, PHisMA is expected to be sensitive to pH in a
biologically relevant range compared to the other pH sensitive
zwitterionic polymers. PHisMA polymer brushes were
synthesized on the gold substrate by surface-initiated photo-
iniferter-mediated polymerization (SI-PIMP). The properties of
PHisMA at different pHs to resist protein absorption and cell
adhesion were studied. The viscoelastic properties and water
uptake of PHisMA hydrogels are also investigated under
1. INTRODUCTION
Over the past 20 years, antifouling materials have attracted
considerable attention from many applications, such as
coating,1 biomedical devices,2−4 drug and gene delivery
carriers,5−9 membranes and protein purifications,10−12 bio-
sensors,13−15 and wound dressings.16 Antifouling materials,
which can respond to environmental stimuli (such as pH, ionic
strength, or temperature), have many advantages over sole-
functional antifouling materials.17−22 Among antifouling
materials, poly(ethylene glycol) (PEG) and its derivatives are
commonly used due to their steric exclusion effect and surface
hydration to repel biomacromolecules.18,23−28 However, PEG is
susceptible to oxidation degradation and likely to lose its
protein repulsive properties at high temperatures.29,30 Due to
their excellent antifouling properties, structure flexibility and
tunable properties, zwitterionic polymers have emerged as a
group of promising ultralow fouling materials.18,29,31 Among
zwitterionic materials, amino acid based polymeric materials
have several advantages due to their unique structures
containing pH sensitive anion (carboxylate) and cations
(primary or secondary amines).32 Moreover, amino acid side
chains can improve the aqueous solubility of polymers and
promote a higher order structure through intra- and interchain
interaction via noncovalent forces such as hydrogen bonding,
hydrophobic stacking and electrostatic interactions.33−38 In
Received: April 15, 2016
Revised: May 31, 2016
© XXXX American Chemical Society
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(DTCA) on the gold-coated SPR chips, the SPR chips were washed
with acetone, isopropanol, and water, cleaned with a UV-ozone cleaner
for 20 min, washed with water and ethanol, and then dried under a
stream of filtered air. Subsequently, SPR chips were soaked in THF
solution containing 0.1 mM photoiniferter at room temperature for 24
h. Prior to the polymerization, the SPR chips were rinsed with THF
and then dried with a stream of filtered air.
2.5. Surface Initiated Photoiniferter-Mediated Polymer-
ization (SI-PIMP). The monomer solution was first prepared by
dissolving 0.5 g of HisMA in 10 mL of PBS at pH 8.5. The solution
was deoxygenated by passing a continuous stream of nitrogen through
the liquid for 20 min. The gold chip with photoiniferter SAM was
placed into a quartz tube under nitrogen protection. After 20 min, the
monomer solution was transferred to a quartz tube using a syringe and
then a stream of nitrogen was purged into the reaction tube for
another 5 min before being removed and covered with aluminum foil.
Subsequently, the quartz tube containing the SPR chip and monomer
solution was irradiated under 302 nm UV light (UVP, Model UVM-
57). A 280 nm cutoff filter was put between the reaction tube and UV
lamp to avoid the cleavage of the thiol−gold bond of the
photoiniferter SAM.41,42 After 2 h, the SPR chip was removed,
washed with PBS, and put into PBS solution overnight before SPR
study.
different pH conditions. Moreover, the chelating capability of
PHisMA hydrogel to high-valent (+2 and +3) metal cations was
determined.
2. EXPERIMENTAL SECTION
2.1. Chemicals. L-Histidine (≥99%), sodium sulfate (≥98%),
acetone (≥99.5%), 2-propanol (≥99.5%), tetrahydrofuran (THF)
(≥99%), 2-hydroxy-4′-(2-hydroxy-ethoxy)-2-methylpropiophenone
(98%), N,N′-methylene-bis(acrylamide) (99%), phosphate buffered
saline (PBS), nickel(II) sulfate hexahydrate (99%), dioxane (≥99.0%),
and lysozyme (from chicken egg white, 53 000 units/mg) were
purchased from Sigma-Aldrich (St. Louis, MO). Methacrylic anhydride
(94%), copper(II) chloride dihydrate (98%), and iron(III) chloride
anhydrous (98%) were purchased from Alfa-Aesar (Ward Hill, MA),
and 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-
044) was purchased from Wako Chemicals USA (Richmond, VA).
Hydrochloric acid (36.5−38%) and ethyl ether (99.0%) were
purchased from EMD Chemicals (Darmstadt, Germany). Ethanol
was purchased from Decon Laboratories, Inc. Pepsin (from porcine
gastric mucosa, 10 635 units/mg) was purchased from Chem-Impex
Int’l. Inc. (Wood Dale, IL).
2.2. Synthesis and Characterization of Histidine Methacry-
lamide and Poly(histidine methacrylamide). Histidine methacry-
lamide (HisMA) was synthesized based on the previously reported
synthetic method.40 L-Histidine (10 g, 64 mmol) was dissolved in 2 N
NaOH (40 mL) aqueous solution and cooled in an ice bath.
Methacrylic anhydride (30 mL, 195 mmol, 3 equiv) was dissolved in
80 mL of dioxane. The methacrylic anhydride solution was added to
the aqueous solution of L-histidine dropwise in a 500 mL three-necked
round-bottom flask under nitrogen protection. During the addition,
the reaction mixture was kept in an ice bath. Then the mixture was
reacted overnight at room temperature. After the reaction, dioxane was
evaporated and hydrochloric acid was added until the solution reached
pH 2. The residue chemicals and byproducts were removed by ether
extraction three times. The pH value of the aqueous solution was
adjusted to 5 using 2 N NaOH. The product was extracted with
ethanol. By this process, L-histidine and NaCl were removed. The
ethanol was evaporated and mixed with an excess of acetone. The
product HisMA precipitated in acetone, and it was then vacuum-dried
overnight. A 10.7 g sample of HisMA was obtained at a yield of 75%.
1H NMR (300 MHz, D2O) 1.83 (s, 3H, CH3C(CH2)H−), 3.01−
2.6. Measurements of Nonspecific Protein Absorption by
SPR. All SPR experiments were performed at 25 °C using a four-
channel SPR sensor (PLASMON-IV, Institute of Photonics and
Electronics, Academy of Science, Czech Republic). The changes of
resonance wavelength were measured by the SPR sensor at a fixed
light incident angle. All buffers were freshly prepared, filtered using 0.2
μm syringe filters, and degassed. The PHisMA-coated SPR chip was
attached to the base of the prism. The refractive index matching fluid
(Cargille) was used as an optical contact between two surfaces. After
first establishing a preabsorptive baseline using PBS at a flow rate of 50
μL/min under ambient temperature, the protein adsorption in the
lysozyme solution (1 mg/mL) and pepsin solution (1 mg/mL) at
different pHs (pH 3, pH 5 and pH 7) was studied. The amount of
adsorbed proteins was calculated from the change in wavelength
before and after protein injection. A 1 nm SPR wavelength shift is
equivalent to 15 ng/cm2 adsorbed protein.20
2.7. Bacteria Attachment. The method for evaluating the
antibacterial efficiency of polymer surfaces was based on a previously
reported method.43,44 E. coli K 12 was first cultured in Luria−Bertani
(LB) medium (20 g/L). The cultures were incubated at 37 °C with
shaking at 200 rpm overnight to reach an optical density of 0.8 at 600
nm. Cell pellets were washed three times with sterile PBS (pH 7.4)
and subsequently suspended in PBS to obtain a final concentration of
109 cells/mL. A 100 μL volume of E. coli suspension was dropped onto
gold-coated glass slides with grafted polymer brushes, which were then
covered with clean microscope slides. The polymer brush surfaces
were preplaced into buffers with different pH values (pH 3, pH 5, and
pH 7) to reach a stable charged state. The samples were stained with
100 μL of LIVE/DEAD BacLight Bacterial Viability assay (Thermal
Scientific, Waltham, MA, USA) solution and then incubated at room
temperature for 1 h. The cover slides were removed, and the samples
were placed in Petri dishes containing 5 mL of buffers with different
pH values at 37 °C with gentle shaking for 1 h in order to wash the
detached bacteria. The amount of attached cells was determined using
an Olympus IX81 fluorescence microscopy with 60× oil lens through
FITC and Cys3 filters. Three samples were analyzed for each gold
substrate separately.
3.28 (m, 2H, −CH2-imidazole), 4.47−4.51 (q, H, −NHCH(COOH)-
CH2−), 5.37−5.58 (m, 2H −CH2C(CH3)−), 7.17 (s, 1H imidazole,
−CCHN), 8.48 (s, 1H imidazole, −NCHNH−).
Poly(histidine methacrylamide) (PHisMA) was synthesized via the
thermally initiated free radical polymerization method. The water-
soluble thermal initiator VA-044 (2,2′-azobis[2-(2-imidazolin-2-yl)-
propane] dihydrochloride) (0.03 g, 0.09 mmol) and HisMA monomer
(2 g, 9 mmol) were dissolved in 20 mL of ultrapure water in a 100 mL
round-bottom flask under nitrogen protection. The polymerization
proceeded for 24 h at 50 °C. After 24 h, the solution was cooled to
room temperature and then dialyzed in water with molecular weight
cutoff of 10 kDa for 2 days. The product was then lyophilized for 2
days. Gel permeation chromatography (GPC) was carried out using an
SEC gel column (Agilent Technologies, Santa Clara, CA) and an
internal refractive index (RI) detector (Waters Corp., Milford, MA).
For PHisMA, running buffer (containing 0.01 M NaH2PO4 and 0.3 M
NaNO3) was used as the eluent at a flow rate of 1 mL/min at 25 °C.
The molecular weight (MW) was 10 986 g/mol.
2.8. Preparation of Hydrogels. Chemically cross-linked hydro-
gels were prepared via photoinitiated free radical polymerization in 1
mL of water at different pH values (pH 3, pH 4, pH 5 and pH 7).
First, 6.68 mg of photoinitiator, 2-hydroxy-4′-(2-hydroxy-ethoxy)-2-
methylpropiophenone, was dissolved in water by sonicating at 40 °C.
Then 11.56 mg of cross-linker, N,N′-methylene-bis(acrylamide), was
added into the initiator solution and sonicated in an ice bath to avoid
gel formation between photoinitiator and cross-linker. After the
solution became clear, 0.336 g of HisMA monomer was added to the
solution. After the filtration by a 0.2 μm filter, the solution was added
2.3. Titration of HisMA and PHisMA. 5 mL of HisMA and
PHisMA solutions at a concentration of 0.1 M were prepared in
deionized water, and the solutions were adjusted to pH 1 using
hydrochloric acid. Then the solutions were titrated against 0.25 N
NaOH solution by adding 20 μL of NaOH solution each time until the
pH reached 13. The change in pH was monitored by a pH meter.
2.4. Preparation of Photoiniferter-Coated Surface Plasmon
Resonance (SPR) Chips. Before the formation of a self-assembled
monolayer (SAM) of the photoiniferter 11-mercaptoundecane-1-[4-
({[(diethylamino)-carbonothioyl]thioethyl}phenyl)carbamate]
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Scheme 1. Synthetic Route of HisMA
1
Figure 1. H NMR spectrum for HisMA.
to the model with 25 mm diameter. The obtained hydrogels were 25
mm in diameter and 2 mm in height.
weighed again. The binding capacity of PHisMA hydrogel (D) to Cu2+,
Ni2+,Ca2+, Mg2+, or Fe3+was calculated using the following equation.
2.9. Rheological Studies. The in situ rheological studies were
used to access the viscoelastic properties of PHisMA hydrogels.
PHisMA hydrogels were equilibrated in the buffer at different pH
values overnight. Measurements were performed at ambient temper-
ature with a frequency of 1 rad/s and a strain of 10%. PHisMA
hydrogels were loaded between 25 mm parallel plate geometry on a
TA Instruments ARES-G2 rheometer. The frequency for different
samples was based on the gel network structure. The storage modulus
and loss modulus were monitored during the measurement process.
2.10. Swelling Ratio Measurement. For swelling ratio studies,
PHisMA hydrogels were made 25 mm in diameter and 2 mm in height
in buffers with different pH values via photopolymerization. After
swelling equilibrium in ultrapure water overnight, the weight of
PHisMA hydrogels was recorded and then the hydrogels were placed
in a freeze-dryer and lyophilized prior to being weighed again. The
swelling ratio, Q, was calculated using the following equation.
D = ((WC − WD)/Mw)/WD
where WC is the dry weight of the hydrogel after chelating with Cu2+,
Ni2+, Ca2+, Mg2+, and Fe3+; WD is the dry weight of PHisMA hydrogel
before the chelation; and Mw is the molecular weight of the
corresponding salt.
3. RESULTS AND DISCUSSION
As shown in Scheme 1, HisMA was synthesized via a one-step
reaction. The chemical structure was characterized by 1H NMR
spectroscopy (Figure 1). Since the pKa of acid/base groups in a
zwitterion determines its charged state under different pH
conditions, the pKa values of the carboxylate and imidazole
groups of HisMA were measured by titration. As shown in
Figure 2, the monomer and polymer titration study revealed
pKa values of 1.82 and 6.12 for the carboxylate and imidazole
groups, respectively, which are consistent with those in
histidine.45At low pH conditions (pH <1.82), both carboxylate
and imidazole groups are protonated, which leads to cationic
HisMA and PHisMA. Between pH 1.82 and pH 6.12, both
carboxylate and imidazole are partially charged. HisMA and
PHisMA have balanced charges, and they have properties of
zwitterionic molecules. At higher pH conditions (pH >6.12),
most carboxylate and imidazole groups are deprotonated, and
HisMA and PHisMA become anionic. In zwitterionic
molecules, the acidity of carboxylate and the basicity of
amine are affected by the distance between carboxylate and
Q = W /WD × 100%
S
where WS is the weight after swelling and WD is the weight after
lyophilizing.
2.11. Metal Ion Chelation Studies. For chelation studies,
PHisMA hydrogels were prepared 8 mm in diameter and 2 mm in
height in ultrapure water. Then, PHisMA hydrogels were lyophilized
and weighed. Subsequently, gels were immersed into ultrapure water
to reach the swelling equilibrium and submerged into 1 M copper(II)
chloride, nickel(II) sulfate, calcium chloride, magnesium sulfate, and
ferric chloride solutions, respectively, to chelate metal ions. After 24 h,
the equilibrated PHisMA hydrogels were placed into ultrapure water
to release unbounded metal ions prior to being lyophilized and
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the adsorption of both proteins on PHisMA polymer brush
surfaces at pH 3 and pH 7 were much higher compared to pH
5, which suggested PHisMA was under different charged states.
At pH 3, the protonation of the imidazole group and partial
protonation of carboxylate switch the polymer surface to be
cationic and the negatively charged pepsin adsorbed more than
that at pH 7, whereas the positively charged lysozyme adsorbed
at pH 3 less than that at pH 7. At pH 7, the deprotonation of
carboxylate and imidazole made the surface anionic and
positively charged lysozyme adsorbed more compared to pH
3. PHisMA polymer brush surfaces showed better protein
resistance at pH 5 with 0.5 ng/cm2 lysozyme and less than 0.3
ng/cm2 pepsin, suggesting a balanced charge state in this pH
range. The results from pepsin and lysozyme absorption
indicate that the PHisMA surface can switch among cationic,
zwitterionic and anionic states by changing the pH of the
solution.
Figure 2. pH titrations of HisMA (solid line) and PHisMA (dashed
line) from pH 1 to 13.
Tunable antimicrobial and antifouling properties are very
useful for medical device coatings and wound dressings. Due to
the distinct surface charges under different pH conditions,
PHisMA was expected to catch cells at low pH conditions and
resist the attachment of cells at an intermediate pH range.
Bacterial cell attachment onto PHisMA polymer brush surfaces
was investigated using E. coli K12 as a model cell. E. coli K12 at
the concentration of 109 cells/mL was incubated with PHisMA
polymer brush surfaces in buffers at different pH values (pH 3,
pH 5 and pH 7) as shown in Figure 3, a very small number of
cells were observed on PHisMA surfaces in pH 5 buffer after
culturing overnight. In contrast, more E. coli K12 cells attached
on PHisMA surfaces in pH 3 and pH 7 buffers. The cell
densities on the surface were (46.24 2.43) × 104, (0.76
0.47) × 104 and (27.59 2.25) × 104 cells/cm2, respectively, at
pH 3, pH 5 and pH 7. PHisMA surface at pH 5 has the best
antifouling property to resist bacterial attachment. PHisMA
surface at pH 7 shows higher bacterial attachment, although
both the surface and E. coli K12 are negatively charged under
this condition. It should be noted that the adhesion/adsorption
of cells/biomolecules to the surface is determined by all
interactions including electrostatic, hydrogen bond and van der
Waals interactions. A similar phenomenon was explained
according to electrostatic interactions and between cells and
surfaces, as described by DLVO theory.46 Our study also
demonstrates that zwitterionic surfaces are by far the most
reliable antifouling surfaces under all tested conditions. The
protein absorption and bacteria attachment results illustrate
that the antifouling/fouling properties of PHisMA polymer
brushes can be tuned over the pH range that is more relevant to
biological conditions.
amine, the density of the charged groups and the substitution
group of the amine.39 It was found that the acidity of
carboxylate increases with the decrease in the carbon spacer
between amine and carboxylate. The charge state of the
molecule significantly affects the interaction of molecules with
solvent, salt, macromolecules, and itself, which subsequently
will change the antifouling, mechanical, and other physical
properties of the material.
In complex biological systems, the property of a material or
device to resist the adsorption of biomacromolecules is critical
for its interaction with cell, service life and performance. To
evaluate antifouling properties under three charged states,
PHisMA polymer brushes on gold substrate were prepared via
surface-initiated photoiniferter-mediated polymerization. The
pH-dependent single protein absorption on PHisMA surfaces
was studied between pH 3 and pH 7 by a surface plasmon
resonance (SPR) sensor. Lysozyme with an isoelectric point
(pI) of 11.35 and pepsin with a pI of 2.2−3.0 were used as the
model proteins. These proteins were chosen as their contrast
charged states in the pH 3−7 range. Lysozyme was positively
charged while pepsin formed the negatively charged counter-
part over the entire pH range in our study. Table 1 shows that
Table 1. Pepsin and Lysozyme Absorption on PHisMA
Surfaces under Different pH Conditions (pH 3, pH 5, and
pH 7)
pH 3
pH 5
pH 7
a
pepsin adsorption (ng/cm2)
lysozyme adsorption (ng/cm2)
8.0
2.5
≪0.3
0.5
0.5
10.0
a
Detecting limit.
Hydrogels are widely used in many biomedical and biotech
applications due to their high water content, tunable and
Figure 3. Bacterial attachment on PHisMA polymer brush surfaces under different pH environments. The scale bar is 50 μm. The cell densities on
the surface were (46.24 2.43) × 104, (0.76 0.47) × 104, and (27.59 2.25) × 104 cells/cm2, respectively, at pH 3, pH 5 and pH 7.
D
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comparable mechanical properties to tissues, biocompatibility,
etc. Many of these properties are determined by the interaction
among polymer chains. Zwitterionic polymers typically show
antielectrolyte properties. Electrolytes/salts screen the attrac-
tive interaction among cations and anions. At the presence of
the electrolytes/salts, the zwitterionic polymer exists in a more
stretched conformation. In contrast, the electrolytes/salts
screen the repulsive interaction among the solely charged side
chains in cationic or anionic polymers, so cationic and anionic
polymers will switch from the stretched state to the random
coils in the presence of the electrolytes/salts. The conforma-
tional change of the polymer affects the properties of the
hydrogels. To have a better understanding of the mechanical
properties of the hydrogel, viscoelastic and swelling properties
of PHisMA hydrogel were studied at different pH conditions.
PHisMA hydrogels were prepared by UV-induced free radical
polymerization. After equilibration in the sodium acetate buffer
at different pHs, the hydrogels were characterized using the
rheological technique. Hydrogels were studied by two
rheological measurements: (1) the strain-sweep measurement
under a certain frequency obtained from the frequency-sweep
experiment suggests that subsequent time sweeps are carried
out in the linear viscoelastic region, and does not influence the
gel network structure; (2) a frequency-sweep measurement
using small strain allows us to determine the equilibrium
modulus under different pH environments.
collapses and aggregates due to interchain and intrachain
electrostatic attractions, which lead to higher cross-linking
density of the hydrogel. For the hydrogel at anionic or cationic
states, PHisMA polymer chains are stretched due to the
repulsive force among charged groups, which results in lower
cross-linking density and viscoelastic properties. The equili-
brium modulus of PHisMA hydrogel at the cationic state is
lower than that at the anionic state. This phenomenon can be
caused by the different cross-linking density caused by a higher
degree of hydrogen bond contributed by uncharged imidazole
groups or the osmotic activities of the different counterions
around the charged groups. The results from rheological studies
point out that zwitterionic polymers have higher equilibrium
modulus and higher cross-linking density owing to stronger
inter- and intrachain interaction, whereas the anionic and
cationic PHisMA show lower viscoelastic properties due to the
inter- and intrachain repulsion.
The swelling ratios of PHisMA hydrogels in buffers at
different pH values (pH 3, pH 4, pH 5, and pH 7) were
consistent with rheological data. As shown in Table 2, the water
uptakes of anionic and cationic PHisMA hydrogels are much
higher than that at the zwitterionic state. The charges within
zwitterionic hydrogels can act as reversible cross-linkers
resulting in variations of the effective network density. The
electrostatic interactions between either opposite charges or
dipole−dipole pairs in zwitterionic hydrogels lead to higher
cross-linking density in the polymer network. Our study
demonstrated that we could control the swelling properties of
the hydrogel by adjusting the pH of the solution. The pH
sensitive hydrogels can potentially be used as pH actuators for
sensors and devices.
Figure 4 shows storage modulus and loss modulus of
PHisMA hydrogels at various pH conditions (pH 3, pH 4, pH 5
The capability of polyelectrolytes to chelate metal ions is
particularly attractive for biofilm management, wound healing
and environmental applications. Since both carboxylate and
imidazole are common chelating groups, we expect that
PHisMA is able to chelate high-valent metal ions. The chelating
capability of PHisMA hydrogel on multi-valent metal ions
(Ca2+, Mg2+,Cu2+, Ni2+ and Fe3+) were investigated in this
study. The amounts of metal ions absorbed by PHisMA were
calculated and are shown in Table 3. The images of PHisMA
Table 3. Binding Capacity of PHisMA Hydrogel to Calcium
Chloride, Magnesium Sulfate, Copper(II) Chloride,
Nickel(II) Sulfate, and Ferric Chloride
cation
D (mmol/g)
Figure 4. Rheological studies of PHisMA hydrogels. Frequency sweep
conducted under 10% strain and 100−0.1 rad/s using 25 mm parallel
plate.
calcium(II)
magnesium(II)
copper(II)
nickel(II)
1.82
3.03
2.86
3.02
2.87
and pH 7). The equilibrium moduli (Table 2) indicate that the
viscoelastic properties of PHisMA hydrogel in the zwitterionic
state are much higher than those in the cationic and anionic
states. In the low ionic strength solution, zwitterionic polymer
iron(III)
hydrogels after chelation with Cu2+, Ni2+ and Fe3+ are displayed
in Figure 5. Among all tested metal ions, PHisMA has strong
binding affinity to +2 and +3 metal ions. Notably, the control of
free metal ions is extremely important for biofilm/infection
management. Of medical importance, biofilm can withstand
attack from the immune system and resist antibiotic/biocide
treatments. The treatment of biofilm is one of the most
important medical and environmental challenges. A previous
study unveiled that magnesium, calcium and iron ions play a
significant role in the development of Pseudomonas aeruginosa
biofilm, which is one of the major species causing hospital
Table 2. Equilibrium Modulus and Swelling Ratio of
PHisMA Hydrogels under Different pH Conditions (pH 3,
pH 4, pH 5, and pH 7)
equilibrium modulus (Pa)
swelling ratio (%)
pH 3
pH 4
pH 5
pH 7
8 000
150 000
60 000
35 000
1259.73
324.12
768.37
1108.96
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Figure 5. Images of PHisMA hydrogels after chelation with (a) nickel(II), (b) iron(III), and (c) copper(II) ions.
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infections, and the removal of the high-valent ions can prevent
biofilm formation and decrease the viability of the biofilm.47
Since PHisMA can chelate multi-valent metal ions, PHisMA
may potentially inhibit biofilm formation by reducing the
amount of the free metal ions in the wound. Further biofilm
formation experiments will be examined in the future.
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4. CONCLUSION
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We synthesized the amino acid based pH-responsive PHisMA
material, which is capable of switching among zwitterionic,
anionic and cationic forms, reducing adsorption of proteins and
inhibiting cell adhesion at its zwitterionic state, carrying tunable
viscoelastic properties, water uptakes and chelation capability to
multi-valent metal ions. Through this study, we established a
better understanding of structure−property relationships of
zwitterionic PHisMA. We were able to obtain the desired
properties by adjusting the pH of the system. This material may
be an excellent candidate for wound treatment, gene delivery,
and membrane and protein purifications, which require
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: gc@uakron.edu. Fax: 330-972-7250. Web: http://
gozips.uakron.edu/~gc/index.html.
Notes
The authors declare no competing financial interest.
(17) Cao, B.; Li, L.; Wu, H.; Tang, Q.; Sun, B.; Dong, H.; Zhe, H.;
Cheng, G. Zwitteration of Dextran: a Facile Route to Integrate
Antifouling, Switchability and Optical Transparencyinto Natural
Polymers. Chem. Commun. 2014, 50, 3234−3237.
(18) Jiang, S.; Cao, Z. Ultralow-Fouling, Functionalizable, and
HydrolyzableZwitterionic Materials andTheir Derivatives forBiological
Applications. Adv. Mater. 2010, 22, 920−932.
ACKNOWLEDGMENTS
■
This work is supported by the U.S National Science
Foundation (ECCS-1200032, DMR-1206923 and DMR-
1454837) and the Natural Science Foundation of China
(NSFC-51528301).
(19) Cheng, G.; Mi, L.; Cao, Z.; Xue, H.; Yu, Q.; Carr, L.; Jiang, S.
Functionalizable and Ultrastable Zwitterionic Nanogels. Langmuir
2010, 26, 6883−6886.
(20) Cheng, G.; Li, G.; Xue, H.; Chen, S.; Bryers, J. D.; Jiang, S.
Zwitterionic Carboxybetaine Polymer Surfaces and Their Resistance to
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Jiang, S. Reversibly Switchable Polymer with Cationic/Zwitterionic/
Anionic Behavior Through Synergistic Protonation and Deprotona-
tion. Chem. Sci. 2014, 5, 200−205.
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