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Supplementary Information
Physical Hydrogels with High Toughness and Viscoelasticity from Polyampholytes
Tao Lin Sun1†, Takayuki Kurokawa2†, Shinya Kuroda1, Abu Bin Ihsan3,
Taigo Akasaki3, Koshiro Sato3, Md. Anamul Haque2, Tasuku Nakajima2, Jian Ping Gong2★
1Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
2Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan
3Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
†These authors contributed equally to this work.
*E-mail: [email protected]
Physical hydrogels composed of polyampholytes demonstrate high toughness and viscoelasticity
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Methods
Synthesis of polyampholyte hydrogels
Polyampholyte hydrogels are synthesized using the one-step random copolymerization of an anionic
monomer and a cationic monomer. All of the chemicals were used as purchased without further
purification. A mixed aqueous solution with the prescribed total ionic monomer concentration Cm (M)
and molar fraction f of the anionic monomer, 0.25 mol% UV initiator, 2-oxoglutaric acid (in relative
to the total monomer molar concentration), and 0.5 M NaCl was poured into in a reaction cell
consisting of a pair of glass plates with a 3 mm spacing and irradiated with 365 nm UV light for 11
hours. After polymerization, the as-prepared gel was immersed in a large amount of water for 1 week
to reach equilibrium and to wash away the residual chemicals. During this process, the mobile
counter-ions of the ionic copolymer are removed from the gel, and the oppositely charged ions on the
copolymer form stable ionic complexes either through intra- or interchain interactions. Fig. 1b and
Supplementary Fig. 6 show the chemical structures of the monomers used in this work. These
cationic and anionic monomers are essentially randomly copolymerized in the polyampholyte
hydrogels. The random structure of P(NaSS-co-MPTC) was confirmed using a 1H-NMR reaction
kinetics study. DN and PAAm hydrogels were synthesized using the method described in reference 8.
Characterization of gels
Swelling measurements. The as-prepared polyampholyte gels formed in glass plates with rectangle
shapes were cut into samples with fixed sizes and then immersed in water and allowed to reach the
equilibrium state. The swelling volume ratio Qv was defined as the ratio of the sample volume at
swelling equilibrium V to that in the as-prepared state V0, Qv = V/V0. The polymer weight fraction
Cpoly (wt%) of the sample was measured by the weight change upon drying using a freeze-drying
process. The swelling of samples in NaCl solution was characterized by the volume ratio of the
equilibrium swollen gel sample in NaCl solution Vsalt to that in water Vwater, Qsalt,water = Vsalt/Vwater. To
achieve adequate precision, three measurements were carried out on samples of different volumes
taken from the same gel.
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Tensile and compressive test. The tensile stress-strain measurements were performed using a
tensile-compressive tester (Tensilon RTC-1310A, Orientec Co.) at a deformation rate of 100 mm/min
in air. The test was carried out on dumbbell-shaped samples with the standard JIS-K6251-7 size (12
mm (L) × 2 mm (d) × 2–3 mm (w)) (Supplementary Fig. 12). For the cyclic tensile test, all of the
experiments were carried out in a water bath to prevent water from evaporating from the samples.
The work of extension at fracture Wb (J/m3), a parameter that characterizes the work required to
fracture the sample per unit volume, was calculated from the area below the tensile stress-strain
curve until fracture. In the compression test, samples with cylinder shapes (~ 15 mm in diameter and
2.5–8 mm in initial thickness) were placed on a metal plate coated with silicon oil to decrease the
friction. The loading velocity was 0.5 mm/min.
Tearing test. The tearing test was performed to characterize the toughness in air using a commercial
test machine (Tensilon RTC-1310A, Orientec Co.). Samples of 2–3 mm (w) in thickness were cut
into the standard JIS-K6252 1/2 sizes (50 mm (L) × 7.5 mm (d); the length of the initial notch is 20
mm) with a gel-cutting machine (Dumb Bell Co., Ltd.)36. The two arms of a test piece were clamped,
and then the upper arm was pulled upward at constant velocity 100 mm/min while the tearing force F
was recorded. The tearing energy T was calculated at a constant tearing force F using the relation T =
2F/w, where w is the thickness of the sample (Supplementary Fig. 13).
Pure shear test. A pure shear test was also used to characterize the toughness, following the method
established in references 18 and 37. Two different samples, notched and unnotched, were used to
measure the tearing energy T. The samples were cut into a rectangular shape with a width of 20 mm
and length 40 mm (a0). The sample thickness was 0.67 mm (b0). An initial notch of 20 mm in length
was cut using a razor blade. The test piece was clamped on two sides, and the distance between the
two clamps was fixed at 8 mm (L0). The upper clamp was pulled upward at constant velocity of 100
mm/min, while the lower clamp was fixed. The force-length curves of the samples were recorded,
and the tearing energy was calculated from T = U(Lc)/(a0 ×b0), where U(Lc) is the work done by the
applied force to the unnotched sample at the critical stretching distance Lc, and Lc is the distance
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between the two clamps when the crack starts to propagate in the notched sample. The onset of the
crack propagation was determined using the movie image recorded by a camera (Supplementary Fig.
14). We have confirmed that the tearing test and the pure shear test gave the consistent values for the
same kind of samples.
Impact test. The shock absorbance ratio was characterized using an impact tester (No.511-D, MYSS
Tester Co.). First, plate-shaped samples with thicknesses of ~3 mm were fixed in the impact tester.
Then, the hammer impacted the sample with a velocity that was determined by the impacting angle.
The impacting velocity (v0) and rebounding velocity (vt) of hammer were calculated from the impact
displacement and time. The shock absorbance ratio R was estimated using the relation R = 1− (vt/v0)2.
The initial impact velocity was fixed at 0.643 m/s.
Rheological test. Rheological tests were performed using an ARES rheometer (advanced rheometric
expansion system, Rheometric Scientific Inc.). A rheological frequency sweep from 0.01 to 15.85 Hz
was performed with a shear strain of 0.5% in the parallel-plates geometry in a temperature range of
0.1–98 °C. The disc-shaped samples with thicknesses of ~ 3 mm and diameters of 15 mm were
adhered to the plates with glue and surrounded by water.
Crack tip observation. In order to observe the stress concentration during the crack growth, the crack
microstructure was frozen using acetone to avoid any stress relaxation, and then the sample was
observed using polarized optical microscopy (Olympus, BH-2).
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1) 1H-NMR reaction kinetics study
9 8 7 6 5 4 3 2 1ppm
a bd ec
5h30min
4h40min
3h40min
2h30min
0min
H2O DMSO
NaSS MPTC
a
f
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0.0 0.5 1.0 1.5 2.0 2.5
0.000
0.008
0.016
0.024
0.032
[MP
TC]
[NaSS]/[MPTC]
r1=1.48r2=0.70
b c
d
0.0 0.5 1.0 1.5 2.0 2.5
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Reaction 1r1=1.45r2=0.713
Reaction 2r1=2.43
r2=0.412
[MP
TC]
[NaSS]/[MPTC]
0.3-0.7 0.52-0.48 0.7-0.3Reaction 3
r1=5.23
r2=0.188
0.0 0.2 0.4 0.6 0.8 1.0
2
4
6
8
10
<N>NaSS
<N>MPTC
<N> N
aSS a
nd <
N> M
PTC
Total monomer conversion, p
Supplementary Figure 1 1H-NMR reaction kinetic study for the copolymerization of NaSS and
MPTC. a, Spectra evolution of reaction mixture in 1H-NMR probe. b, Molar concentration of MPTC,
[MPTC], vs the ratio [NaSS]/[MPTC] from the 1H-NMR analysis of the 3 reaction systems with
different NaSS molar fraction 0.7 (reaction 1), 0.52 (reaction 2) and 0.3 (reaction 3). c, Corrected
global molar concentration of MPTC, [MPTC], vs the ratio [NaSS]/[MPTC] using the treatment
proposed in reference 38. d, The instantaneous number-average sequence length of monomer NaSS
<N> NaSS and MPTC <N> MPTC versus the total monomer conversion p for 0.52:0.48 composition.
The dashed line is the predicted curve of sequence length change with monomer conversion.
The random structure of polyampholyte hydrogel P(NaSS-co-MPTC) was confirmed using a
1H-NMR reaction kinetics study. In order to determine the sequence length distribution of the
copolymer P(NaSS-co-MPTC), the reactivity ratios r1 and r2 are needed to calculate from the
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monomer conversion against reaction time, where the r1 and r2 are defined as the ratio of rate
constants of home-propagation reactions to cross-propagation reactions for NaSS and MPTC,
respectively. The detailed method used here is described in the reference 38 and 39.
The copolymerization of anionic monomer NaSS and cationic monomer MPTC was carried out in
D2O and the weighted dimethyl sulfoxide (DMSO) was used as external standard substitute. Three
kinds of mixed aqueous solution with total monomer concentration 0.1 mol/L, the NaSS molar
fraction (0.3, 0.52 and 0.7), 0.25 mol% UV initiator, 2-oxoglutaric acid (in a concentration relative to
the total monomer concentration), and 0.5 mol/L NaCl were performed in the glass tubes under the
irradiation of UV light. To study the monomer conversion, we taked the samples from the reaction
system and transfered to a shaded place to quench the polymer reaction each several minutes. The
concentration of unreacted monomers remaining in solution was determined by the integral area ratio
of 1H-NMR signals which were detected by a 400MHz NMR system, that is, the vinylic protons of
the monomers (6.8, 5.9 and 5.4ppm for NaSS, 5.7 and 5.5 ppm for MPTC) versus DMSO protons
(2.8ppm) which corresponds to the symbols c, a, b, d, e and f in the spectra (Supplementary Fig. 1a).
Finally, we obtained the monomer conversion spectra against reaction time.
From the conversion of the monomer, we determined the reactivity ratios using the integrated form
of the copolymerization equation (terminal model) for the three reactions38, as described in
Supplementary Fig. 1b. The reactivity ratios are r1 = 5.23, r2 = 0.188 for reaction 3, r1 = 2.43, r2 =
0.412 for reaction 2 and r1 = 1.45, r2 = 0.713 for reaction 1 by fitting the terminal model,
corresponding to the composition of NaSS 0.3, 0.52 and 0.7, respectively. The obtained different
reaction ratios are due to the sample preparation and integration which perturb the measurement to
result in the large error. To overcome this, we obtained the combined data by multiplying the inverse
shift factors 21
1
[ ] 10[ ]
reaction
reaction
MPTCQMPTC
at the common ratio [ ] 1.011[ ]
NaSSMPTC
(the final and initial
monomer ratio for these two reactions) with data of reaction 2 and 32 1
2
[ ] 31.04[ ]
reaction
reaction
MPTCQ QMPTC
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at the ratio [ ] 0.303[ ]
NaSSMPTC
with data of reaction 3, respectively, as indicated in Supplementary
Fig. 1c. Finally, the obtained reactivity ratio is r1 = 1.48 and r2 = 0.70. Furthermore, the apparent
rate constants of propagation reactions of NaSS and MPTC at the composition of fNaSS = 0.52 are
0.00711/min and 0.00272/min, respectively, according to the relationship between the monomer
conversion and reaction time. Then we can predict the required time for full conversion of total
monomers.
The instantaneous number-average sequence length of NaSS monomer, <N> NaSS, and MPTC
monomer, <N> MPTC, can be expressed with the total monomers conversion point p according to the
Mayo-Lewis theory39. The results are shown in Supplementary Fig.1d. We predicted the sequence
length with the monomer conversion at high monomer conversion p (> 0.8) from the relationship
between the conversion of total monomers and reaction time when the apparent rate constants of
propagation reactions of monomers are known.
According to these results, we can assume the mechanism of the supramolecular hydrogels network
formation. The polymerization begins with the incorporation of NaSS molecules with a few MPTC
molecules added due to the difference of reactivity ratios (r1 = 1.48 and r2 = 0.70). At total monomer
conversion p = 0, the determination of <N>NaSS = 2.5 and <N>MPTC = 1.7 can be understood that, as
an average of the incorporation to the growing polymer chains, a sequence of three NaSS molecules
follows by one MPTC molecules. At p = 0.8, then <N>NaSS = 1.3 and <N>MPTC = 5.1, a sequence of
one NaSS molecule would follow by 5 MPTC molecules. At higher convention (> 0.9), the polymer
chains will grow with a block sequence of MPTC molecules. This polymerization process leads to
the inhomogeneities of the network structure. As a result, we assume that during the dialysis process
the NaSS rich segments (formed at the beginning of polymerization) and MPTC rich segments
(formed at the end of polymerization) would form the strong ionic complex structure, serving as
permanent cross-linking points, while other parts lead to the weak ionic complex, behaving as
reversible sacrificial bonds.
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2) Effect of charge ratio
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
100
101
102
Swelling volume ratio
Young's modulus
Fracture stress
NaSS molar fraction in feed, f
Sw
ellin
g vo
lum
e ra
tio, Q
v
10-2
10-1
100 Fracture stress,
b (MP
a)
Deswelling
Young's m
odulus, ESwelling
Supplementary Figure 2 NaSS molar fraction effects on the swelling volume ratio Qv, Young’s
modulus E, and the compressive fracture stress σb of the hydrogels P(NaSS-co-MPTC).
We denote the samples using the symbol Cm-f-x and the names of the copolymers, where Cm (mol/L)
is total molar concentration of monomers, f is the anion molar charge fraction, and x (mol %) is the
molar ratio of the chemical cross-linker N, N′-methylenebisacrylamide (MBAA) in relative to Cm in
the precursor solution.
Supplementary Fig. 2 shows the effect of the charge fraction (f) on the swelling volume ratio Qv,
Young’s modulus E, and the compressive fracture stress σb of the hydrogels P(NaSS-co-MPTC) Cm
-f-4 synthesized with different NaSS molar fractions f in the feed, where the total molar
concentration Cm is fixed at 0.875 mol/L with a chemical cross-linker (x = 4 mol %). Here, Qv is
defined as Qv = V/V0, where V and V0 are the volumes of the samples after full swelling in pure water
and in the as-prepared state, respectively.
Near the charge balance point (f = 0.48 ~ 0.53), the gels shrink in water (Qv < 1) relative to their
as-prepared state, indicating that the Coulomb attraction prevails over the repulsion and the polymer
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chains collapse. In the regions with sufficient charge imbalance (f < 0.48 or f > 0.53), on the other
hand, the gels swell (Qv > 1), indicating that the Coulomb repulsion prevails and the polymer
segments elongate. The shrinking of the gels around the charge balance point is accompanied by
dramatic increases in the modulus (E) and fracture stress (σb). Thus, the optimized f value in the feed,
at which Qv reaches a minimum and E and σb reach maximums, is 0.52, which is close to the charge
balance point.
The true charge ratio of P(NaSS-co-MPTC) Cm-f-4 (the total molar concentration is fixed at
0.875mol/L) was studied using elemental analysis (Supplementary Table1), which revealed that ftrue
= 0.48 for the sample of f = 0.52 in feed, which is slightly different from the ideal stoichiometric
ratio of 1:1. This indicates that the gel with complete charge balance (f = 0.5) is not in the most stable
state. The attractive ion pairs cannot achieve close approach, probably because the restriction of the
polymer chain conformation frustrates the electrostatic effect23.
Supplementary Table 1 The weight percentage of elements in various polyampholyte hydrogels
P(NaSS-co-MPTC) 0.875-f-4 through element analysis.
f wt % (C) wt % (H) wt % (N) wt % (S) wt % (Cl) ftrue
0.450 53.27 9.00 7.97 6.74 2.00 0.412
0.480 54.53 8.54 7.84 7.16 1.10 0.438
0.495 55.46 8.35 7.90 7.56 0.76 0.444
0.500 55.42 8.25 7.62 7.85 0.45 0.458
0.505 53.04 8.34 7.30 7.79 0.37 0.465
0.520 57.23 7.90 7.75 8.25 0.00 0.475
0.550 54.08 7.91 6.88 8.34 0.00 0.511
*f is the anion charge fraction in feed and ftrue is the true anion charge fraction in gel determined by
element analysis.
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3) Molecular weight
To discuss how the entanglements of polymer chains contribute to the toughness of the hydrogels, we
have tried to estimate the molecular weight of the polymer. The P(NaSS-co-MPTC) 2.1-0.52
hydrogels dissolved in 4 M saline solution at high temperature (> 50°C) after 2 days, and we can
obtain homogeneous aqueous solution. It is difficult to accurately estimate the molecular weight Mw
of such a kind of random polyampholyte using Gel Permeation Chromatography (GPC), so we
estimated Mw, roughly, from the overlapping concentration C* of the polymer solution where the
viscosity increased dramatically. The overlap concentration determined by the viscosity in 4 M saline
solution was C*~ 30 g/L, corresponding to the repeat unit ~ 0.15 mol/L. C* is related to the repeat
unit size a (~ 0.3 nm), the average degree of polymerization N, and the coil size of a polymer chain R
as:
3~
43A
NCN R
Assuming that the polymer is in the Θ solvent, 1/2~R aN . So we have
23
3~ ( )4 A
NN a C
Where, the NA is Avogadro constant (6.02×1023 mol-1). As a result, the degree of polymerization is N
~10000, and the corresponding molecular weight is around ~ 2×106 g/mol. This value falls in the
common range of radical polymerization.
Commonly, the entanglement concentration Ce is about several times above the overlap
concentration. As shown in Fig. 2a, the gel phase appears at Cm ~ 0.7 mol/L. This value is 5 times the
value of C*, consistent with the entanglement concentration Ce. This supports the argument that the
gel phase starts to appear above the entanglement concentration.
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4) Deswelling-induced complex formation as revealed by cyclic testing
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Stre
ss (M
Pa)
Strain (mm/mm)
As-prepared gel
Equilibrium gel
a
b
Supplementary Figure 3 Schematics of polyampholyte hydrogel in the as-prepared state and
equilibrium state in water. a, Illustration of the dialysis process from the as-prepared state to the
equilibrium state of the gel. b, Cyclic stress-strain curves of the as-prepared and water-equilibrium
gel P(NaSS-co-MPTC) 2.1-0.52. The equilibrium state of the gel is measured in water, while the
as-prepared state of the gel is measured in air.
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5) Lattice model for calculating the electrostatic interaction
Supplementary Figure 4 Schematic lattice model of rod-like polymer chains for calculating the
electrostatic energy. a, The cationic (red) and anionic (blue) groups are alternately distributed along
the polymer chains. The distance between the cationic and anionic groups is the same. The rod
consists of two concentric cylinders of inner radius r and outer radius R, which correspond to the
radius of bare and hydrated macroions, respectively. b, Projection of the top part of the profile in (a).
We use the lattice model shown in Supplementary Fig. 4 to estimate the polymer concentration
dependence of the strength of polyampholyte hydrogels. From this lattice model, the polymer volume
fraction, defined as the volume of dry gel divided by the volume of wet gel, can be expressed as 2
2dry
wet
V rV R
, (1)
while the polymer concentration Cpoly is related to the polymer volume fraction,
~ drypoly
wet
VC
V (2)
By substituting Eq. (1) into Eq. (2), we can get 2
2~polyrCR
(3)
Since the size of the bare macroions r is fixed, Eq. (3) becomes
0.5~ polyR C (4)
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According to the electrostatic interaction theory40, the ion association energy Eion is given by the
equation 2
0
~4 2
Aion poly
r
N eE CR
(5)
Here, NA is the Avogadro constant, e is the charge of an electron, R is the hydrated radius of the ion,
and 0 and r are the vacuum permittivity and relative permittivity of water, respectively. Then, Eq. (5)
is simply described by the scaling relation,
~ polyion
CE
R (6)
From Eq. (4) and Eq. (6), the power relations between the ionic interaction and the concentration of
the polymer is derived as
1.5~ion polyE C (7)
This scaling law is in agreement with the experimentally observed relation between the tearing
energy T and the true polymer concentration Cpoly,
1.8~ polyT C (8)
This agreement quantitatively illustrates the effect of the ionic interactions on the strength and
toughness of the polyampholyte hydrogels.
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6) Solvent-induced shape memory
Writing Erasing
37s 6min
a b f
c d e
Supplementary Figure 5 Shape memory behaviour of polyampholyte hydrogel. The as-prepared
hydrogel P(NaSS-co-MPTC) 2.1-0.52 with its initial straight shape (a) is deformed into a spiral
shape that can be ‘written’ by immersing the sample in water (b). When the sample is stretched, the
spiral shape is deformed to a straight shape (c); however, it recovers its spiral shape automatically
after the force is removed (d, e, b). The full recovery process takes about 20 min in water at 20 °C.
The spiral shape can be erased in 0.5 M NaCl solution, which causes the sample to return to its initial
straight shape (f).
Since the ion complexes serve as cross-linking points and lock the polymer chain conformations, we
can ‘write’ any desired shape to the polyampholyte hydrogels during the ion complex formation
process in water and ‘erase’ the shape by dissociating the ion complexes in NaCl solution. As shown
in Supplementary Fig. 5, when we deform an as-prepared hydrogel from its initial straight shape
(Supplementary Fig. 5a) into a spiral shape and then immerse it in water, the spiral shape is
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memorized (Supplementary Fig. 5b, write process). After that, even if the gel is forced to deform to
the straight shape, it automatically returns to the spiral shape (Supplementary Fig. 5c, d, e).
Furthermore, when we immerse the spiral shape gel in 0.5M NaCl solution, the memorized spiral
shape is erased and the gel recovers to its initial straight shape (Supplementary Fig. 5f, erase process).
In principle, the write and erase processes can be repeated many times. A softening temperature Ts ~
48.2°C (Supplementary Fig. 7c) is observed, and the written spiral shape is memorized either below
(25°C) or above (75°C, Supplementary Movie 3) this Ts in water, which confirms that the shape
memory effect is solvent-induced. This effect is different from the shape memory effects of most
polymers, which are based on the glass transition temperature of the polymer41-42.
7) More polyampholyte hydrogels systems and chemical structure effect
MTAC 4-VPC
Supplementary Figure 6 Chemical structures of cationic monomers for the polyampholyte
hydrogels. Methacrylatoethyl trimethyl ammonium chloride (MTAC) and 4-vinylpyridine chloride
(4-VPC).
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Supplementary Table 2 Structural features and mechanical properties of various polyampholyte
hydrogels.
Hydrogels Composition
Cm-f*
cw
(wt%)
E
(MPa)
σb (MPa) εb
(m/m)
Wb
(MJ/m3)
tanδ R
(%)
Ts
(°C)
poly
amph
olyt
e hy
drog
els P(NaSS-co-MPTC)(1) 2.1-0.52 54.3 2.05 1.82 7.42 7.12 0.24 76.6 48.2
P(NaSS-co-DMAEA-Q)(2) 2.0-0.52 52.6 0.10 0.14 15.5 1.31 0.58 95.5 17.3-
P(NaSS-co-4-VPC)(3) 2.0-0.5 61.1 7.92 1.25 6.42 5.05 0.24 77.1 54.2
P(NaSS-co-MTAC) 2.0-0.5 52.3 0.042 0.33 13.77 2.14 0.56 87.9 47.1
Com
mon
hyd
roge
ls
PAAm
Single network
2-4(4) 84.1 0.092 0.067 0.65 0.024 0.044 - -
PAMPS/PAAm
double network
1-4/2-0.01(5) 86.0 0.12 1.04 12.38 8.94 0.02 61.57 -
* Cm and f represent the total ionic monomers concentration (mol/L) and the charge fraction of the
anionic monomer, respectively, in the precursor solution used to synthesize the gels. The parameters
cw, E, σb, εb, Wb, tanδ, and R are the water content, Young’s modulus, fracture stress, fracture strain,
work of extension at fracture, loss factor (10 Hz and strain 0.5%), and shock-absorbing ratio,
respectively, at room temperature. Ts is the softening temperature determined by the peak of loss
factor of the gels.
(1)(2) The optimal compositions at the minimum swelling ratio Qv used were to obtain the strongest
mechanical properties, while other samples were synthesized at the stoichiometric ratio in feed. (3)
The gels were synthesized from 4-vinylpyridine (4-VP), and then the as-prepared samples were
immersed in 0.5 M HCl to ionize the weak bases 4-VP to their charged forms 4-VPC, before the
immersion in water. (4) The abbreviated symbol 2-4 refers to 2 M AAm and 4 mol% MBAA in the
precursor solution of the gel. (5) The 1-4/2-0.01 notation represents 1 M AMPS and 4 mol% MBAA
for the first network and 2 M AAm and 0.01 mol% MBAA for the second network in the precursor
solution.
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0.32 0.40 0.48 0.56 0.64
0
1
2
3
4
Deswelling
P(NaSS-co-MPTC) P(NaSS-co-DMAEA-Q) P(AMPS-co-DMAEA-Q)
Swel
ling
volu
me
ratio
, Qv
Anionic monomer fraction in feed, f
Swelling
a b
c
48.217.3
0 3 6 9 12 15 180.0
0.4
0.8
1.2
1.6
2.0
P(NaSS-co-DMAEA-Q)52.6% water content
Stre
ss (M
Pa)
Strain (mm/mm)
53.9% water contentP(NaSS-co-MPTC)
0 20 40 60 80 1000.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
P(NaSS-co-MPTC) P(NaSS-co-DMAEA-Q)
Loss
fact
or, T
an
Temperature, T (oC)
Supplementary Figure 7 Effect of chemical structure of the ionic monomers on the behaviours of
hydrogels. a, Relationship between the swelling volume ratio Qv and the anionic monomer molar
fraction f of the 3 sets of gels prepared at a formulation of 0.875-f-4. All the gels deswell around their
charge balance points (f ~ 0.5). b, Tensile behaviours of the two physical polyampholyte hydrogels
prepared at the f = 0.52 where their Qv reaches a minimum: P(NaSS-co-MPTC) 2.0-0.52,
P(NaSS-co-DMAEA-Q) 2.0-0.52. c, Temperature dependence of the loss factor (tanδ) of
P(NaSS-co-MPTC) 2.1-0.52 and P(NaSS-co-DMAEA-Q) 2.0-0.52 at 10 Hz and 0.5% strain. The
temperature at which tanδ reaches the maximum corresponds to the softening temperature Ts, which
is indicated by the numbers in the figure. The experiments were performed in water.
This one-step approach to synthesizing polyampholyte hydrogels with multiple specific mechanical
properties is quite general and can be applied to various combinations of oppositely charged
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monomers. The Young’s modulus and the viscoelastic behaviours of the synthesized gels depend
strongly on the specific chemical structure, especially the hydrophobicity of the monomers. To
demonstrate these effects, we used two additional monomers, cationic methyl chloride quarternised
N,N-dimethylamino ethylacrylate (DMAEA-Q) and anionic 2-acrylamido-2-methylpropanesulfonic
acid (AMPS), as shown in Fig. 1b. Using the ionic monomer combinations, we compare the
behaviours of three series of hydrogels with hydrophobicities in the order of P(NaSS-co-MPTC) >
P(NaSS-co-DMAEA-Q) > P(AMPS-co-DMAEA-Q). Their swelling behaviours of the three series of
chemically crosslinked gels at various charge fractions f are shown in Supplementary Fig. 7a. The
most hydrophilic P(AMPS-co-DMAEA-Q) series exhibits a sharper V-shape than the other two
series; that is, a slight deviation from the charge balance point destroys the ion complex. This
indicates that the hydrophobicity has a synergistic effect in stabilizing the ionic interaction23.
The linear polyampholytes were prepared at the f where their Qv reaches a minimum in
Supplementary Fig. 7, f = 0.49 for P(AMPS-co-DMAEA-Q) and f = 0.52 for P(NaSS-co-MPTC) and
P(NaSS-co-DMAEA-Q) ). No physical hydrogel is formed from the most hydrophilic combination of
AMPS and DMAEA-Q. In contrast, tough physical gels are formed in the relatively hydrophobic
combination of NaSS and DMAEA-Q, the same like the combination of MPTC and NaSS. As shown
in Supplementary Fig. 7b, the most hydrophobic physical hydrogels, P(NaSS-co-MPTC) 2.0-0.52,
which contains 53.9 wt% water, is very tough and shows clear yielding, a high strength (σb = 1.7
MPa), and a relatively large extensibility (εb = 730%). The less hydrophobic physical hydrogels,
P(NaSS-co-DMAEA-Q) 2.0-0.52, which contains 52.6% water, is very ductile, showing a large
extensibility (εb = 1550%). Thus, the more hydrophobic the gel, the stronger the ion bond is.
The less hydrophobic physical hydrogels, P(NaSS-co-DMAEA-Q) 2.0-0.52, shows almost perfect
self-healing that is better than that of the more hydrophobic and rigid sample, P(NaSS-co-MPTC)
2.1-0.52. After healing for 24 h at room temperature, the two cut surfaces completely merged
together and the healing efficiency reached as high as ~ 99%, as shown in Fig. 3f.
The strength of the ion bonds also dramatically influences the viscoelasticity and, therefore, the
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damping behaviours of the physical hydrogels. For example, at room temperature, the less
hydrophobic P(NaSS-co-DMAEA-Q) 2.0-0.52 with a weak ion complex shows a shock-absorbance
ratio R as high as 95.5%, while the hydrophobic P(NaSS-co-MPTC) 2.1-0.52 with a strong ion
complex exhibits an R of 76.6% (Supplementary Table 2). The viscoelastic and shock-absorption
features of the polyampholyte hydrogels are presented in the movies showing the vibration and
rebound experiment, in which the elastic double-network gel is used as the reference (Supplementary
Movies 4 and 5).
8) Structure analysis
-200 -100 0 100 200 300-4x104
-3x104
-2x104
-1x104
0
Wet gel
Hea
t flo
w (u
w)
Temperature (oC)
Dry gel
4 6 8 10 12 14 16 18 20 22
50
100
150
200
250
Wet gelInte
nsity
(a.u
.)
q (nm-1)
Dry gel
a b
Supplementary Figure 8 Structure analysis by WAXS and DSC. a, WAXS spectra of wet and dry
P(NaSS-co-MPTC) 2.1-0.52 samples. The WAXS patterns were obtained by a Rigaku X-ray
crystallograph under Cu radiation (λ = 0.15418 nm). The measurement was carried out using an
X-ray generator with a voltage of 40 kV and a current of 20 mA. The specimen-to-detector distance
was 250 mm, and the exposure time was 5 min. b, DSC scanning for wet and dry P(NaSS-co-MPTC)
2.1-0.52 samples performed at a heating rate of 10 °C/min from -150 °C to 300 °C. The two peaks
(around 0 °C and 120 °C) in the DSC curves are assigned to the melting point and boiling point of
water in the polymer, respectively. Thermal analysis was performed using a differential scanning
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calorimeter (Model DSC22, SII Nano Technology Inc.) connected to a thermal analysis system
(model, SSC 5100).
We have attempted to analyse the structure of the hydrogels P(NaSS-co-MPTC) 2.1-0.52 using
several methods. The gel shows no X-ray diffraction peaks in the wide-angle X-ray scattering
(WAXS) range, while a broad peak at q ~ 12.7nm-1, corresponding to a characteristic length of 0.49
nm, appears for the dried sample (Supplementary Fig. 8a). Furthermore, no thermal melting peaks
from the ion complex structure appear in differential scanning calorimetry (DSC) analysis either for
the hydrogels or for the dried sample (Supplementary Fig. 8b). These results indicate that the
polyampholyte hydrogels are amorphous with no crystalline structure, which is different from the
behaviour of ionomers that form crystalline domains43.
9) Rheological results
a b
10-10 10-8 10-6 10-4 10-2 100 102 104 106104
105
106
107
64.1C 72.1C 80.1C 88.1C 95C
G' G'' Tan0.1C 8.1C 16.1C 24.1C 32.1C 40.1C 48.1C 56.1C
Frequency, (Hz)
Stor
age
mod
ulus
, G'
Loss
mod
ulus
, G''
(Pa)
0.2
0.3
0.4
0.5
0.6
Loss factor, Tan
2.6 2.8 3.0 3.2 3.4 3.6 3.8-15
-10
-5
0
5
10
15
Ea= 308kJ/mol
ln a
T
1/T (10-3K-1)
Ea= 71kJ/mol
Supplementary Figure 9 Dynamic mechanical behaviours of the polyampholyte hydrogels
P(NaSS-co-MPTC) 2.1-0.52. a, Frequency (ω) dependence of the storage modulus G′, loss modulus
G″, and loss factor tanδ of polyampholyte hydrogel. The measurements were performed from 0.01 to
15.8 Hz at a shear strain of 0.5% at different temperatures from 0.1 °C to 95 °C, and the results were
obtained by performing classical time-temperature superposition shifts at a reference temperature of
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24.1 °C. b, Arrhenius plot depicting the temperature dependence of the shift factors for the sample.
The apparent activation energy values were calculated from the slope of the curve. These values are
smaller than the covalent bond dissociation energy Ec-c ~ 347 kJ/mol, which ensures the preferential
dissociation of the ion complexes under deformation.
The dynamic behaviours of the polyampholyte hydrogel at different temperatures and frequencies
follow the principle of time-temperature superposition well. Supplementary Fig. 9a shows the master
curves of G′, G″, and tanδ for the polyampholyte hydrogel at a reference temperature of 24.1 °C. It is
noted that G′ is larger than G″ over the whole frequency range from 10-7 to 106 Hz, indicating that
the sample, even without any chemical cross-linking, is always in the gel state with predominantly
elastic properties. The longest relaxation time, estimated as the reciprocal of the frequency at which
the storage modulus reaches a plateau at about 6×104 Pa, is about 6×106 s at 24.1°C. The apparent
activation energy Ea is obtained from the Arrhenius equation, /aE RTTa Ae , where aT is the shift
factor, R is the ideal gas constant, and A is a constant44. The temperature dependence of the shift
factor aT shows that the activation energy of the gel varies over a wide range, 71–308 kJ/mol, as
shown in Supplementary Fig. 9b, corresponding to 29–124 kBT at room temperature. All these results
indicate a wide distribution of strengths of the ion associations, which corresponds to the random
structure obtained from the radical polymerization of the sample. The upper range of the activation
energy is less than but close to the covalent bond dissociation energy, Ec-c ~ 347 kJ/mol (~ 140 kBT).
This explains why a rigid and tough gel is observed even without any covalent cross-linker in this
system. We also found that the activation energy Ea of the less hydrophobic system hydrogel
P(NaSS-co-DMAEA-Q) 2.0-0.52 has a narrower distribution of Ea (112–248 kJ/mol) than the more
hydrophobic system P(NaSS-co-MPTC) 2.1-0.52.
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10) Biocompatibility
In order to evaluate the biocompatibility and anti-biofouling properties of polyampholyte
hydrogels P(NaSS-co-MPTC), Chinese hamster lung fibroblast cells (JCRB0603:V79) and RAW
264.7 macrophages (Primary Cell Co. Ltd. Japan) were used to perform the cytotoxicity test and
adhesion test, respectively.
Cytotoxicity test of polyampholytes hydrogels using V79 cells
1.0 1.5 2.0 2.5 3.0 3.520
40
60
80
100
120 Positive RM-C
Col
ony
form
atio
n ra
te (%
)
Concentration (ug/ml)
a b
0.1 1 10 1000
30
60
90
120
150
Col
ony
form
atio
n ra
te (%
)
Concentration (%)
Gel 2.0-0.525 Gel 1.9-0.525 Gel 1.5-0.525 Positive RM-A Positive RM-B
Supplementary Figure 10 Cytotoxicity Test of substances using V79 cells. a, Colony formation rate
of polyampholyte physical hydrogels Poly(NaSS-co-MPTC) 2.0-0.525, 1.9-0.525 and 1.5-0.525,
positive control substance (RM-A) and positive control substance (RM-B) with different
concentration extracted from these substances. b, Colony formation rate of positive control substance
(RM-C) with different concentration extracted from the substance.
This study was conducted to investigate the cytotoxic effects of the extracts from polyampolyte
hydrogel using Chinese hamster lung fibroblast cells (JCRB0603:V79) by the colony formation
method, referring to the standard method: ISO 10993-5: 2009 (E) Biological evaluation of medical
devices – Part 5: Tests for in vitro cytotoxicity; and ISO 10993-12: 2007 (E) Biological evaluation of
medical devices – Part 12: Sample preparation and reference materials.
Physical polyampholyte hydrogels Poly(NaSS-co-MPTC) with compositions 2.0-0.525, 1.9-0.525,
and 1.5-0.525 were used to investigate the cytotoxic effects toward the Chinese hamster lung
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fibroblast cells (JCRB0603: V79); V79 cells obtained from the Department of Cancer Cell Research,
Institute of Medical Science, University of Tokyo on March 9, 1994 were used. High-density
polyethylene film (negative RM) was used as the negative reference material (abbreviation: negative
RM) that shows no cytotoxicity. Polyurethane film containing 0.1% zinc diethyldithiocarbamate was
used as the positive reference material A (abbreviation: positive RM-A) that shows moderate
cytotoxicity. Polyurethane film containing 0.25% zinc dibutyldithiocarbamate was used as the
positive reference material B (abbreviation: positive RM-B) that shows weak cytotoxicity. Zinc
dibutyldithiocarbamate directly dissolved in dimethyl sulfoxide was used as the positive reference
material C (abbreviation: positive RM-C). These controls were purchased from Hatano Research
Institute, Food and Drug Safety Center. MEM culture medium containing 5% fetal bovine serum
(abbreviation: M05 culture medium) were prepared by mixing the components at the following ratios.
Firstly, Eagle MEM culture medium (9.4 g; containing kanamycin and phenol red) was dissolved in
Japanese Pharmacopoeia water with a total volume of 1 L for injection. Secondly, the sodium
bicarbonate solution was added to the sterilized mixture by autoclaving to adjust the pH to 7.2 to 7.4.
Thirdly, MEM nonessential amino acid solution (0.09 mmol/L), sodium pyruvate solution (0.11 g/L),
L-glutamine (0.292 g/L), and fetal bovine serum (inactivated at 56ºC for 30 minutes, 5%) were added
to the system to achieve the final composition. The sterilized polyampholyte hydrogels by
autoclaving (120ºC, 20 minutes), approximately 2 (thickness) × 15 (diameter) mm, were put in a
borosilicate glass medium bottle. Then the culture medium (10 ml/g) was added making the hydrogel
reach the equilibrium state after 24 hours in a shaking incubator which was set under the condition of
37ºC, amplitude 70 mm, and 100 rpm. The culture medium after the extraction from the substrates
(100% extraction) were collected and diluted to the prescribed concentrations as the testing solutions.
The extracts obtained from the controls A and B were also diluted by the same method. Controls C
was diluted with dimethyl sulfoxide to obtain the prescribed concentrations. The resulting solutions
were used to test. Each test group for substrates with different concentration was assayed in
triplicate.
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Cells in a growth phase were treated with 0.02% EDTA-0.25% trypsin (0.5M EDTA, 2.5% trypsin,)
and harvested, and then suspended in the culture medium to obtain a cell density of 50 cells/mL
counted by a hemocytometer. The suspension was dispensed to a 12-well multiwell plate (FALCON)
at the volume of 1 mL (50 cells)/well which was placed in the CO2 incubator at 5.0% CO2 and 37.0
ºC. 1 mL/well of the test solution was added to the plate for culturing for 6 days before discarding of
the initial culturing medium. After culturing, the culture medium for the test solution in the well was
discarded, and then the cells on the substrate were rinsed with Ca2+ and Mg2+ free Dulbecco’s
phosphate buffer and fixed in methanol for approximately 5 minutes. Then the cells were stained
with 5% Giemsa solution for approximately 10 to 15 minutes. Each well was observed with a
stereoscopic microscope (SZ61TRC-C-D, Olympus Corporation) to count the colonies consisting of
50 cells or more. Any well obviously showing a decrease in the colony size (a decrease in the number
of cells per colony) was also recorded.
For each test series, the colony formation rate in each well was calculated regarding the mean
number of colonies in the corresponding control group (0% or 0 µg/mL: blank) as 100%. When the
colony formation rate decreased to 50% or less, the IC50 (concentration that inhibited the colony
formation rate to 50% of the control mean value) value was calculated using regression analysis after
logarithmic conversion of concentrations based on the concentration-response relationship. The
cytotoxicity of the test substrate is classified as shown in the Supplementary Table 3 with reference
to the Basic Principles of Biological Safety Evaluation Required for Application for Approval to
Market Medical Devices, MHLW Notification 0301 No. 20, Office of Medical Devices Evaluation,
Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health,
Labour and Welfare, Japan, March 1, 2012.
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Supplementary Table 3 Classification of cytotoxicity
Degree of cytotoxicity (IC50 value) Classification of cytotoxicity
100% or more No cytotoxicity, or very weak
cytotoxicity
Weaker than the positive RM-B Weak cytotoxicity
Stronger than the positive RM-B and weaker than the
positive RM-A Moderate cytotoxicity
Stronger than the positive RM-A Severe cytotoxicity
The colony formation rates of the polyampholyte hydrogels Poly(NaSS-co-MPTC) 2.0-0.525,
1.9-0.525, 1.5-0.525, positive control RM-A, RM-B, and RM-C are shown in Supplementary Fig.
10a and b. The extracts from the negative RM do not inhibit the colony formation of the cells.
However, the colony formation is inhibited at concentration 2% or more of the extracts from the
positive RM-A, at concentration of 60% or more of the extracts from the positive RM-B, and at
concentration of 3µg/ml or more of the extracts from the positive RM-C, respectively. The IC50
values calculated from the colony formation rates are the concentration of 1.42%, 50.6% and 3.05
µg/ml for the positive RM-A, positive RM-B and positive RM-C, respectively. For polyampholyte
hydrogels 2.0-0.525, 1.9-0.525, 1.5-0.525, no inhibition effect on colony formation phenomenon are
observed during the concentration of 0% ~ 100%, which indicate the nontoxicity of polyampholyte
hydrogels towards the V79 cells (IC50~ 100% or more) according to the degree of cytotoxicity in
Supplementary Table 3.
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Cytotoxicity test and adhesion test of polyampholytes hydrogels using macrophages
104
105
106
722
Num
ber o
f cel
ls p
er c
m3
Time (h)
Control of live cells Control of dead cells Hydrogels of live cells Hydrogels of dead cells
a
TCPS
P(NaSS-co-MPTC) gel 2P(NaSS-co-MPTC) gel 1
PAAm gel
70um
b
0
50
100
150
200
00 1 1.43 4.2 9 2.81 1.4
182 17
24
Num
ber o
f cel
ls p
er m
m2 TCPS
PAAm gel P(NaSS-co-MPTC) gel 1 P(NaSS-co-MPTC) gel 2
2Time (h)
101 9.5
d
c
72 hours
70um
2 hours
Supplementary Figure 11 The behaviors of macrophages on the different substrates. a, Number
density of live and dead macrophages in solution after the cells were cultured on the hydrogel
P(NaSS-co-MPTC) 1.8-0.525 and on tissue culture polystyrene (TCPS) for 2h and 72h. b,
Morphology of macrophages after culturing on TCPS for 2h and 72h in the presence of
polyampholyte hydrogel P(NaSS-co-MPTC) 1.8-0.525. c, Morphology of macrophages adhered on
TCPS, PAAm hydrogel, P(NaSS-co-MPTC) gel 1, and P(NaSS-co-MPTC) gel 2 after culturing for
24h. Red arrows indicate macrophage adhered on the surface of substrates. d, Number density of
macrophages adhered on the substrates after culturing for 2h and 24h.
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In order to evaluate the anti-biofouling properties of polyampholyte hydrogels P(NaSS-co-MPTC),
RAW 264.7 macrophages (Primary Cell Co. Ltd. Japan) were used to perform the cytotoxicity test,
including the direct and indirect contact tests45-46, and adhesion test since that macrophages are
highly adhesive cells responsible for immune response to implant materials.
For the direct test, the cells are cultured on the surface of the hydrogel while for the indirect test,
cells were cultured on the tissue culture polystyrene (TCPS) in the presence of hydrogels.
The sterilized hydrogel samples with the disc-shape of 15mm in diameter for the cell culture were
immersed in HEPES buffer solution to reach the swollen equilibrium state for one week by
continuously exchanging the solution. The morphology of cells on the substrate surfaces were
observed under the phase contrast microscope (OLYMPUS CKX31, Japan) equipped with a digital
camera. The macrophages were diluted in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum and seeded on the substrate surfaces in a 5% CO2
humidified atmosphere at 37ºC.
Cytotoxicity test
Direct contact test Firstly, the cells were cultured on the surface of the physical polyampholyte
hydrogel P(NaSS-co-MPTC) 1.8-0.525, and the TCPS was used as a control. The non-adherent cells
were washed away from the hydrogels using PBS buffer solution while the trypsin was used to
remove the cells adhered to the TCPS substrate. Then the collected cell suspension was mixed with
trypan blue. The live and dead cells were counted by the hemocytometer under the microscopy. The
initial seeded cell density was about 5.0×104 and 1.8×104 cells/cm3 for the live cells and dead cells,
respectively. As shown in Supplementary Fig. 11a, the number density of live and dead macrophages
increase from 5.0×104 and 1.8×104 (2h) cells/cm3 to 6.8×105 and 1.8×105 (after 72h culturing)
cells/cm3, respectively, when the cells were cultured on the P(NaSS-co-MPTC) 1.8-0.525. On the
TCPS control, the value changes from 5.0×104 and 1.8×104 (2h) cells/cm3 to 2.2×106 and 1.0×106
(after 72h culturing) cells/cm3, respectively. The hydrogel shows higher ratio of live cells to dead
cells (3.8) than that on TCPS control (2.8), which demonstrate the nontoxicity of the polyampholyte
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hydrogels. Polyampholyte hydrogels of other compositions also exhibit the same non-toxic tendency
(data are not shown).
Indirect contact test In order to further confirm the nontoxicity of polyampholyte hydrogels
toward macrophages, the cells were cultured on the TCPS in the presence of sample
P(NaSS-co-MPTC) 1.8-0.525. The initial seeded cell density was about 6.8×104 cells/cm3.
Supplementary Fig. 11b shows the morphology of macrophages on TCPS after culturing for 2 h and
72h in the presence of P(NaSS-co-MPTC) 1.8-0.525. Comparing with the initial seeding of the cells,
the cell numbers increased after 72h culturing, indicating the proliferation of the cells. This result
also indicates the non-toxicity of polyampholyte hydrogel P(NaSS-co-MPTC) towards the
macrophages.
Adhesion test
For the cell adhesion test, we used hydrogels P(NaSS-co-MPTC) 2.1-0.52 with 0.1 mol% and 0.3
mol% chemical crosslinker (coded as P(NaSS-co-MPTC) gel 1 and P(NaSS-co-MPTC) gel 2,
respectively) to facilitate the direct optical observation due to their transparent features. We also used
PAAm hydrogel (synthesized from 1 M AAm and 4 mol% MBAA) as a hydrophilic control. About
5.0×104 cells/cm3 cells were seeded on each of these samples, and after 2h and 24 h, the
non-adherent cells were washed away from the substrates using PBS buffer solution. Supplementary
Fig. 11c shows the optical images of macrophages on these substrates. A large number of
macrophages adhere on the hydrophobic surface of TCPS while a small amount of macrophages
adhere on the hydrophilic surface of PAAm gel. However, almost no cell adheres on the surfaces of
the two P(NaSS-co-MPTC) hydrogels. The number of cell adhered on the surfaces, determined from
three images of each sample, also confirms this, as shown in Supplementary Fig. 11d. The number of
adhered macrophages on the surface of TCPS and PAAm gel increase with the culture time from
101±9.5 (2h) to 182±17.0 cells/mm2 (24h), and from 1±1.4 (2h) to 9±2.8 cells/mm2 (24h),
respectively, while, there is no cell adhesion on the P(NaSS-co-MPTC) hydrogel 1 and the number of
adhered macrophage slightly decreases from 3±4.2 (2h) to 1±1.4 cells/mm2 (24h) on
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P(NaSS-co-MPTC) hydrogel 2. These results indicate that the polyampholyte hydrogels have
excellent anti-fouling properties against microphages. The anti-biofouling behavior of the
polyampholyte gels is analogous to zwitterion polymers that usually show anti-biofouling
properties47-49.
The cytotoxicity test and adhesion test demonstrate that the polyampholyte hydrogels have excellent
biocompatibility and anti-biofouling properties.
11) Mechanical test
Tensile test
Supplementary Figure 12 Geometry of tensile test sample. Sample thickness w = 2-3 mm.
Fracture test
In order to obtain the accurate tearing energy of these polyampholyte hydrogels P(NaSS-co-MPTC)
with relatively high modulus, we use two models to determine this, including tearing test and pure
shear test.
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0 20 40 60 80 1000
1
2
3
4
5
6
Forc
e, F
(N)
Streching distance, L (mm)
c
a b
Supplementary Figure 13 Tearing test to determine the tearing energy. a, Geometry of tearing test
sample. Sample thickness w = 2-3 mm. b, Experimental picture of the tearing test. c, A typical
force-extension curve of tearing test for polyampholyte hydrogel P(NaSS-co-MPTC) 2.1-0.52.
Tearing test For the tearing test, the method to determine the tearing energy was introduced in
reference 36. As shown by the constant stretching force in Supplementary Fig. 13b, a steady state of
crack propagation is obtained (Supplementary Fig. 13c). The tearing energy is calculated from the
constant stretching force F as T=2F/w. T is ~ 3950 J/m2 for P(NaSS-co-MPTC) 2.1-0.52.
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a
c
Unnotched sample Notched sample
F
F
F
F
b
0 10 20 30 40 500
10
20
30
40
Notched sample
Unnotched sample
Forc
e, F
(N)
Streching distance, L (mm)
Lc
Supplementary Figure 14 Pure shear test to determine the tearing energy. a, Geometry of notched
sample for pure shear test. Sample thickness b0 = 0.67 mm. b, Experimental photo image of the pure
shear test. For both the unnotched sample (left) and notched sample (right), the upper clamp was
pulled upward at constant velocity of 100 mm/min from their initial distance (L0 = 8mm) between the
two clamps, while the lower clamp was fixed. The force-length curves of the samples were recorded.
c, Force-extension curves of the unnotched (wine) and notched (olive) samples of polyampholyte
hydrogel P(NaSS-co-MPTC) 2.1-0.52. The yellow area is the work U(Lc) done by the applied force
to the unnotched sample at the critical stretching distance Lc that the notched sample start to
propagate the crack. The tearing energy is calculated as T = U(Lc)/(a0×b0).
Pure shear test For the pure shear test, the method to determine this was established in reference
18 and 37 (Supplementary Fig. 14). The calculated tearing energy T of the gel P(NaSS-co-MPTC)
2.1-0.52 is ~ 4350J/m2 which is almost consistent with the value 3950 J/m2 obtained from the tearing
test.
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For very soft samples, only pure shear test was performed successfully. For example, the tearing
energy T of the gel P(NaSS-co-DMAEA-Q) 2.0-0.52 by using pure shear test is ~ 3670J/m2.
References
38. Aguilar, M. R., Gallardo, A., Fernández, M. D. & San Roman, J. In situ quantitative 1H NMR
monitoring of monomer consumption: a simple and fast way of estimating reactivity ratios.
Macromolecules 35, 2036-2041 (2002).
39. Thévenot, C., Khoukh, A., Reynaud, S., Desbrières, J. & Grassl, B. Kinetic aspects, rheological
properties and mechanoelectrical effects of hydrogels composed of polyacrylamide and polystyrene
nanoparticles. Soft Matter 3, 437-447 (2007).
40. English, A. E. et al. Equilibrium swelling properties of polyampholytic hydrogels. J. Chem. Phys.
104, 8713-8720 (1996).
41. Lendlein, A. & Kelch, S. Shape-memory polymers. Angew. Chem. Int. Ed. 41, 2034-2057 (2002).
42. Yakacki, C. M. et al. Strong, tailored, biocompatible shape-memory polymer networks. Adv.
Funct. Mater. 18, 2428-2435 (2008).
43. Quiram, D. J., Register, R. A. & Ryan, A. J. Crystallization and ionic associations in
semicrystalline ionomers. Macromolecules 31, 1432-1435 (1998).
44. Henderson, K. J. & Shull, K. R. Effects of solvent composition on the assembly and relaxation of
triblock copolymer-based polyelectrolyte gels. Macromolecules 45, 1631-1635 (2012).
45. Hsiue, G. H., Hsu, S. H., Yang, C. C., Lee, S. H. & Yang, I. K. Preparation of controlled release
ophthalmic drops, for glaucoma therapy using thermosensitive poly-N-isopropylacrylamide.
Biomaterials 23, 457-462 (2002).
46. Lopes, C. M. A. & Felisberti, M. I. Mechanical behaviour and biocompatibility of
poly(1-vinyl-2-pyrrolidinone)-gelatin IPN hydrogels. Biomaterials 24, 1279-1284 (2003).
47. Cheng, G. et al. Zwitterionic carboxybetaine polymer surfaces and their resistance to long-term
biofilm formation. Biomaterials 30, 5234-5240 (2009).
NATURE MATERIALS | www.nature.com/naturematerials 33
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48. Ladd, J., Zhang, Z., Chen, S., Hower, J. C. & Jiang, S. Zwitterionic polymers exhibiting high
resistance to nonspecific protein adsorption from human serum and plasma. Biomacromolecules 9,
1357-1361 (2008).
49. Jiang, S. Y. & Cao, Z. Q. Ultralow-fouling, functionalizable, and hydrolyzable zwitterionic
materials and their derivatives for biological applications. Adv. Mater. 22, 920-932 (2010).
Supplementary Movie 1
A movie showing that a virgin polyampholyte hydrogel, P(NaSS-co-MPTC) 2.1-0.52, clamped
in a tensile setting, sustains a load of 2 kgf.
Supplementary Movie 2
A movie showing that the self-healing polyampholyte hydrogel, P(NaSS-co-MPTC) 2.1-0.52,
clamped in a tensile setting, sustains a load of 0.2 kgf.
Supplementary Movie 3
A movie showing the quick recovery process of the polyampholyte hydrogel,
P(NaSS-co-MPTC) 2.1-0.52 from the straight shape to the spiral shape when we remove the load in
75 °C water.
Supplementary Movie 4
A movie showing the different viscoelastic behaviours of polyampholyte hydrogel,
P(NaSS-co-MPTC) 2.1-0.52 (left) and DN gel (right). The samples are fixed with the metal clamp to
prevent slipping. When the samples are released from similar deformations, the viscoelastic
polyampholyte hydrogel returns back slowly and the deformation energy is dissipated by the internal
friction, while the purely elastic DN gel shows multiple vibrations. DN gel: PAMPS/PAAm with the
composition shown in Supplementary Table 2.
Supplementary Movie 5
A move showing the different shock-absorbing behaviours of the polyampholyte hydrogel (left)
34 NATURE MATERIALS | www.nature.com/naturematerials
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Page 35
and DN hydrogel (right). The two gel balls are allowed to fall from the same height to the
surface of a hard wooden block with the same velocity. Although the polyampholyte hydrogel
has a much higher stiffness (E = 2.05 MPa) than the DN hydrogel (E = 0.12 MPa), it shows only
a small rebound, indicating its strong viscoelastic nature and good shock absorption
characteristics, while the DN gels rebounds multiple times due to its pure elastic nature. The
samples used have the same compositions as those in Supplementary Movie 4.
NATURE MATERIALS | www.nature.com/naturematerials 35
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