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polymers
Article
Studies of the Sulfonated Hydrogenated
Styrene–Isoprene–Styrene Block Copolymer and Its Surface
Properties, Cytotoxicity, and Platelet-Contacting Characteristics
Bin-Hong Tsai 1, Tse-An Lin 1, Chi-Hui Cheng 2,* and Jui-Che Lin 1, *
Citation: Tsai, B.-H.; Lin, T.-A.;
Cheng, C.-H.; Lin, J.-C. Studies of the
Sulfonated Hydrogenated
Styrene–Isoprene–Styrene Block
Copolymer and Its Surface Properties,
Cytotoxicity, and Platelet-Contacting
Characteristics. Polymers 2021,13,
235. https://doi.org/10.3390/polym
13020235
Received: 21 November 2020
Accepted: 10 January 2021
Published: 12 January 2021
Publisher’s Note: MDPI stays neu-
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Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
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ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan;
colintsai0324@gmail.com (B.-H.T.); s7161s7161@gmail.com (T.-A.L.)
2Department of Pediatrics, College of Medicine, Chang Gung University, Chang Gung Memorial Hospital,
Taoyuan 33305, Taiwan
*Correspondence: pedneph.cheng@msa.hinet.net (C.-H.C.); jclin@mail.ncku.edu.tw (J.-C.L.)
Abstract:
Styrenic thermoplastic elastomers (TPEs) consist of styrenic blocks. They are connected with
other soft segments by a covalent linkage and are widely used in human life. However, in biomedical
applications, TPEs need to be chemically hydrogenated in advance to enhance their properties such
as strong UV/ozone resistance and thermal-oxidative stability. In this study, films composed of
sulfonated hydrogenated TPEs were evaluated. Hydrogenated tert-butyl styrene–styrene–isoprene
block copolymers were synthesized and selectively sulfonated to different degrees by reaction with
acetyl sulfate. By controlling the ratio of the hydrogenated tert-butyl styrene–styrene–isoprene block
copolymer and acetyl sulfate, sulfonated films were optimized to demonstrate sufficient mechanical
integrity in water as well as good biocompatibility. The thermal plastic sulfonated films were found
to be free of cytotoxicity and platelet-compatible and could be potential candidates in biomedical
film applications such as wound dressings.
Keywords: sulfonation; TPE; hydrogenated SEPS; wound dressing; cytotoxicity
1. Introduction
Compared with natural rubbers, thermoplastic elastomers (TPEs) have different at-
tributes: allergen-free, low extractability, ease of sterilization, softness, and clarity for
biomedical applications. Among these TPEs, styrene–isoprene–styrene (SIS) triblock
copolymers have lower hardness, which is suitable for medical tubing or food package
film applications [
1
]. As SIS contains unsaturated carbon–carbon double bonds in the main
chain of the central rubber block, hydrogenation is required to improve its thermal and
light stability when used in the biomedical field. After hydrogenation, SIS converted to
styrene-(ethylene–propylene)-styrene (SEPS) with improved chemical and physical proper-
ties, and these factors have resulted in hydrogenated styrenic block copolymers becoming
widely used medical plastics despite the higher cost [2].
Following the use of SEPS in medical tubing, films, or stoppers, biocompatibility
has increasingly become a vital factor, especially in tissue-contacting applications. It is
generally accepted that material hydrophilicity is among the most important biocom-
patible attributes; thus, surface modification with hydrophilic functional groups such as
hydroxyl or sulfonic is commonly investigated. A common strategy used to improve the
hydrophilicity of TPE is to apply high-intensity UV/ozone or oxidants to activate the
TPE for further surface-grafting processes [
3
–
6
]. Studies have addressed the TPE surface
patterning of hydrophilic polymer brushes and architectural construction are also effi-
cient ways to fabricate biocompatible and bioactive surfaces [
7
,
8
]. In previous studies,
grafting with sulfonic groups on biomaterial has been considered an effective method
to improve biocompatibility [
9
–
14
], and most research has only focused on surface-level
Polymers 2021,13, 235. https://doi.org/10.3390/polym13020235 https://www.mdpi.com/journal/polymers
Polymers 2021,13, 235 2 of 13
modifications [
15
,
16
]. However, regarding soft SEPS, few investigations have addressed
the issue of losing structural integrity during swelling in a water solution after harsh
oxidative chemical treatments [
17
]. Therefore, the balance between surface hydrophobicity
and structural integrity has become challenging in the development of SEPS biomedi-
cal applications. Recent developments of a new hydrogenated styrenic block copolymer
(HSBC) compound regarding water treatment could have led to a new SEPS approach in
the biomedical field. To improve the hydrophilicity of HSBCs without losing their physical
integrity, Kraton LCC developed a controllable sulfonated pentablock styrenic copolymer
(s-PBC), commercialized as Nexar
TM
. Functionalization with sulfonic groups in Nexar
TM
allows this polymer to achieve an excellent balance between various applications, mainly
due to its block molecular structure and functionalization with sulfonic hydrophilicity
and mechanical stability [
18
]. The unreacted tert-butyl functional end group prohibits the
resulting block copolymers from dissolving in water, and sufficient mechanical strength
of swollen copolymer films is also maintained [
19
]. Until now, this methodology has only
been applied to the industrial water treatment field; to the best of our knowledge, no one
has investigated the biocompatibility of sulfonated HSBCs.
In our previous work, we found that the sulfonation of blends of polypropylene/tert-
butyl SEPS demonstrated physical stability as well as biocompatible performance in terms
of mechanical intactness, low platelet adhesion, and being free of cytotoxicity [
20
]. We
also observed that the mechanical property of blends of sulfonated PP/tert-butyl SEPS
with different mixing ratios was brittle, and the brittleness of blends of sulfonated PP/tert-
butyl SEPS film can be reduced dramatically with the aid of mineral oil as an additional
plasticizer. The aim of the present work is to systematically study the effect of the sul-
fonation degree of a neat tert-butyl SEPS copolymer to form biocompatible elastomers
without any plasticizer additive. A triblock copolymer, SIS, was successfully polymerized
via anionic polymerization. The hard segment of SIS was composed of specific ratios of
styrene and tert-butyl styrene to control the degree of sulfonation. SIS is an unsaturated
block copolymer with carbon–carbon double bonds in the molecular chain, leading to low
stability under high sterilization temperature or UV radiation. To overcome this issue, the
SIS triblock copolymer was treated with the hydrogenation process, saturating the existing
double bonds and giving the resulting styrene-ethylene–propylene-styrene (SEPS) triblock
copolymer better thermal properties such as temperature resistance.
After the hydrogenation process, a selective sulfonation process was conducted, intro-
ducing the hydrophilic sulfonic functional groups into the SEPS triblock copolymer. The
degree of sulfonation of SEPS was controlled as a function of the ratio of the tert-butyl
functional group and acetyl sulfate. The sulfonated SEPS films demonstrated a promis-
ing balance of hydrophilicity and structural integrity, which remained physically intact
in an aqueous solution. Finally, the cytotoxicity and platelet-contacting results suggest
that sulfonated SEPS (i.e., sulfonated hydrogenated SIS) exhibits great potential as future
candidates for wound-dressing or tissue-engineering applications.
2. Materials and Methods
2.1. Materials
All reagents used were of analytical grade and carefully demoistured. The essential
materials used in this experiment, which were styrene, 4-tert-butyl styrene, and isoprene,
were obtained from Alfa Aesar (Loughborough, UK). The N-butyllithium solution, cobalt
2-ethylhexanoate solution, and acetic anhydride were supplied by Sigma-Aldrich (St. Louis,
MO, USA). Cyclohexane (J. T. Baker, Allentown, PA, USA), sulfuric acid (J. T. Baker, USA),
1,2-dichloroethane (Alfa Aesar), and triethylaluminum were used as received. Bovine
serum albumin (BSA), sodium dodecyl sulfate (SDS), and phosphate-buffered solution
(PBS, 0.1 mol/L, pH = 7.4) were provided by Thermo Fisher (Waltham, MA, USA).
Polymers 2021,13, 235 3 of 13
2.2. Analytical Methods
1
H NMR analysis of all the samples was recorded on a Bruker AV-500 (Berlin, German)
with CDCl3 as the solvent and tetramethylsilane as an external reference. All the spectra
were referenced at
δ
= 7.25 ppm for 1 H to CDCl
3
. FTIR spectra were recorded with a Varian
630-IR instrument (Palo Alto, CA, USA) with a resolution of 4 cm
−1
in the transmission
mode, in which the samples were cast onto a KBr disk from a solution of the elastomer
dissolved in chloroform. Gel permeation chromatography (GPC) was obtained using a
Waters 150-C ALC/GPC instrument (Millipore, Burlington, MA, USA) equipped with a set
of ViscoGEL I-Series Columns. The surface morphology of sample films was investigated
using a JEOL JSM-6700F HR-FESEM (Tokyo, Japan). The polymeric samples in the form of
thin films, before analysis, were coated with gold in a sputter coater to achieve a conducting
surface and were analyzed at an accelerated voltage (potential) of 10 kV.
2.3. Synthesis of the SIS Triblock Copolymer
A series of SIS triblock copolymers, named S5T5, S6T4, S7T3, S8T2, and S9T1, were
prepared in brief according to the process described in Scheme 1. The feed ratio of the two
monomers styrene and 4-tert-butyl styrene was altered for different copolymers, and the
amount of isoprene was fixed (Table 1).
Polymers 2021, 13, x FOR PEER REVIEW 3 of 13
ceived. Bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), and phosphate-buff-
ered solution (PBS, 0.1 mol/L, pH = 7.4) were provided by Thermo Fisher (Waltham, MA,
USA).
2.2. Analytical Methods
1H NMR analysis of all the samples was recorded on a Bruker AV-500 (Berlin, Ger-
man) with CDCl3 as the solvent and tetramethylsilane as an external reference. All the
spectra were referenced at δ = 7.25 ppm for 1 H to CDCl3. FTIR spectra were recorded with
a Varian 630-IR instrument (Palo Alto, CA, USA) with a resolution of 4 cm–1 in the trans-
mission mode, in which the samples were cast onto a KBr disk from a solution of the elas-
tomer dissolved in chloroform. Gel permeation chromatography (GPC) was obtained us-
ing a Waters 150‐C ALC/GPC instrument (Millipore, Burlington, MA, USA) equipped
with a set of ViscoGEL I-Series Columns. The surface morphology of sample films was
investigated using a JEOL JSM-6700F HR-FESEM (Tokyo, Japan). The polymeric samples
in the form of thin films, before analysis, were coated with gold in a sputter coater to
achieve a conducting surface and were analyzed at an accelerated voltage (potential) of
10 kV.
2.3. Synthesis of the SIS Triblock Copolymer
A series of SIS triblock copolymers, named S5T5, S6T4, S7T3, S8T2, and S9T1, were
prepared in brief according to the process described in Scheme 1. The feed ratio of the two
monomers styrene and 4-tert-butyl styrene was altered for different copolymers, and the
amount of isoprene was fixed (Table 1).
Scheme 1. The synthesis process of styrene-(ethylene–propylene)-styrene (SEPS) and sulfonated SEPS.
In a 250 mL round-bottomed flask, a specific amount of styrene and 4-tert-butyl sty-
rene was dissolved in 80 mL of cyclohexane at room temperature. Great attention must be
taken when conducting moisture-sensitive anionic polymerization. A 0.3 g amount of tert-
BuLi dissolved in 10 mL of cyclohexane was added dropwise with a syringe over a time
of 1 min. The reaction mixture was maintained at 53 °C , and the reaction was continued
Scheme 1. The synthesis process of styrene-(ethylene–propylene)-styrene (SEPS) and sulfonated SEPS.
In a 250 mL round-bottomed flask, a specific amount of styrene and 4-tert-butyl styrene
was dissolved in 80 mL of cyclohexane at room temperature. Great attention must be taken
when conducting moisture-sensitive anionic polymerization. A 0.3 g amount of tert-BuLi
dissolved in 10 mL of cyclohexane was added dropwise with a syringe over a time of
1 min. The reaction mixture was maintained at 53
◦
C, and the reaction was continued for
12 h under constant stirring until the solution turned dark red. A 10 g amount of isoprene
then was added to the reaction mixture and kept in the reaction for 5 h, after which the
same specific amount of styrene and 4-tert-butyl styrene was added for an additional 12 h
reaction and precipitated in 1 L of a methanol/acetone co-solvent for the termination of the
reaction. The precipitated polymer was washed several times with methanol. The polymer
was then dried in a desiccator under vacuum at room temperature.
Polymers 2021,13, 235 4 of 13
2.4. Hydrogenation of the SIS Block Copolymer
SIS copolymers were hydrogenated according to previous procedures [
21
–
24
] with
some modifications. The hydrogenation process was conducted under an argon atmo-
sphere, and all Schlenk flasks were filled with argon after high-temperature heating. A
triethyl aluminum/cobalt 2-ethyl hexanoate complex was used as a homogeneous hy-
drogenation catalyst. Firstly, a Schlenk flask was charged with 20 mL of the cobalt 2-
ethylhexanoate cyclohexane solution (1.34 mmol) followed by cooling in a liquid nitrogen
bath. Secondly, the cobalt 2-ethylhexanoate cyclohexane solution was degassed by a freeze–
pump–thaw cycle two times and injected with 4.8 mL of triethyl aluminum via syringe for
the reaction for 1 h until the solution turned from purple-blue to dark blue.
Thirdly, an argon-purged reactor was loaded with cyclohexane (140 mL) and SIS (6 g)
and stirred until the SIS was fully dissolved. Later, 15 mL of a catalytic complex was
added into the reactor, which was then closed and purged with argon. The reactor was
heated to 60
◦
C and purged with hydrogen, and the hydrogen pressure was increased up
to 6 bar. Six hours later, the catalyst was deactivated by the addition of a citric acid solution
(50 mmol/L). The copolymer was precipitated in acetone/ethanol, washed and redissolved
in cyclohexane, reprecipitated, and dried under vacuum conditions for the final prepared
SEPS block copolymer.
2.5. Sulfonation of the SEPS Block Copolymer
Acetic anhydride (7.63 mL) and 1,2-dichloroethane (39.57 mL) were placed in a two-
necked-bottomed flask at 10
◦
C, followed by the addition of sulfuric acid (15 mL) to obtain
acetyl sulfate. Hydrogenated SIS, the SEPS (2.5 g), was dissolved in 1,2-dichloroethane and
acetyl sulfate (3 mL) was added. The sulfonation reaction was carried out in an oil bath at 50
◦
C
for 2.5 h. The sulfonated copolymer was dropped into boiling water, giving the crude product.
Finally, the degree of sulfonation was determined by titration. In brief, 0.1 g of dehydrated
sulfonated polymer was dissolved in about 10 mL of a toluene/methanol mixture (9:1 vol). A
standard 0.1 N solution of sodium potassium was first diluted 10 times with methanol and
used to titrate the polymer solution, with phenolphthalein as an indicator.
2.6. Streaming Potential
The streaming potential of the flat substrate was determined by the SurPass Electro-
Kinetic analyzer (Anton Paar KG, Graz, Austria). To mimic the physiological conditions,
phosphate-buffered saline (PBS) at pH 7.4 was used as the electrolyte solution. When the
electrolyte solution flowed over the flat substrates under moderate pressure, the streaming
potential was measured by two Ag/AgCl electrodes, placed at the inlet and outlet of the
fluid cell.
2.7. Cytotoxicity Assay
The cytotoxicity evaluation of the SEPS (hydrogenated SIS) films was processed ac-
cording to the ISO10993-5 and ISO-10993-12 standard testing methods. NIH-3T3 fibroblast
cells were used for the biocompatibility assay on these SEPS substrates. Briefly, the NIH
3T3 fibroblast cells were seeded in a 24-well plate at a density of 6
×
10
4
cells/well and
incubated in Dulbecco’s Modified Eagle’s medium (DMEM). The SEPS films were cut to
8 mm thick disks (~1.3 g each) and immersed in a 24-well culture plate with DMEM for
24 h. After 24 h, the culture medium for the NIH 3T3 fibroblast cells was replaced by the
medium obtained from the SEPS incubation/extraction. The cells were further incubated
for another 24 h, and the number of viable cells was determined using an automated cell
counter (TC20, Bio-Rad, Hercules, CA, USA). Briefly, the cells were trypsinized to form
cell suspensions and mixed with trypan blue (0.4%, Gibco, USA) at a volume ratio of 1:1.
The well-mixed solution was added to the cell counting slide, and the viable cell number
was calculated. For the direct-contact cytotoxicity assay, the NIH 3T3 fibroblast cells were
seeded in a 24-well plate, in which SEPS films with a film thickness of about 1 mm (~0.16 g
Polymers 2021,13, 235 5 of 13
each) were preinserted into each well at a density of 6
×
10
4
cells/well and incubated in
DMEM for 24 h.
2.8. In Vitro Platelet Adhesion Test
The sulfonated and nonsulfonated films were placed in a tissue culture plate and
incubated with Hepes–Tyrode’s solution for 1 h before use. The fresh platelet-rich plasma
(PRP) was obtained from the local Tainan Blood Donation Center. The Hepes–Tyrode’s
solution was removed, and 5 mL of the PRP solution was introduced on each film and
incubated for 1 h at 37
◦
C. The adhered platelets on the films were washed with Hepes–
Tyrode’s solution three times and fixed by the Hepes solution containing 2% (v/v) of
glutaraldehyde for 30 min. Finally, the films were dehydrated in ascending ethanol/water
mixtures (25 vol %, 50 vol %, 75 vol %, 100 vol % ethanol) for 3 min during each step.
Finally, the substrates were dried with CO
2
critical point drying and were immediately
sputter-coated with Au for further SEM morphological analyses. The number of adhered
platelets on the films was calculated from several SEM pictures of the same film at a
magnification of 3000
×
. The number of activated (i.e., not in a round shape) adhered
platelets was also examined [25,26].
3. Results
3.1. Material Characterization
In this study, the acronym “S
x
T
y
” denotes the nomenclature of copolymers prepared
from styrene (S) and 4-tert-butyl styrene (T); x and y stand for the weight ratio of styrene
and 4-tert-butyl styrene, respectively (Table 1). In order to obtain a well-defined polymeric
structure, anionic polymerization was selected for the synthesis of a specific ratio of
styrene/4-tert-butyl styrene with isoprene [27].
Table 1. Composition of different SIS copolymers.
S5T5 S6T4 S7T3 S8T2 S9T1
Styrene feeding weight (g) 1.0 1.2 1.4 1.6 1.8
tert-butyl styrene (tbs) feeding weight (g)
1.0 0.8 0.6 0.4 0.2
Isoprene feeding weight (g) 5.0 5.0 5.0 5.0 5.0
Styrene/tbs (experimental weight ratio)
a0.67 0.79 0.91 1.05 1.16
Styrenic block (theoretical mole %) 0.18 0.18 0.19 0.20 0.20
Styrenic block (experimental mole %) a0.39 0.36 0.37 0.37 0.34
1,4 isoprene/3,4 isoprene (molar ratio) a15.45 14.02 13.37 14.24 15.05
aCalculated from 1H NMR.
The NMR spectra of each block of copolymers with different feed weight ratios of
styrene and 4-tert-butyl styrene are shown in Figure 1. The
1
H NMR spectra of SIS showed
characteristic signals of different protons at
δ
values of 7.05–6.57 ppm (aromatic protons
on phenyl rings), 5.1 ppm (vinyl protons of the 1,4-isoprene unit), and 4.7 ppm (vinyl
protons of the 3,4-isoprene unit). A 1.85–2.10 ppm range addressed the methylene proton
of the 1,4-isoprene and 3,4-isoprene units, and the successful protection of the 4-tert-butyl
group was confirmed by the existence of the characteristic signal of the 4-tert-butyl group
at 1.3 ppm [
28
]. The atomic ratio of each block was calculated by the integrated area of
the characteristic peak [
29
,
30
]. Based on Figure 1, the peak integration area of 1.3 ppm
decreased with the increment of the styrene ratio from 5:5 to 9:1. Specific proton chemical
shifts showed that the reaction was well controlled. The molar ratio of each block was
calculated using Equation (1):
styrene : tert −butyl styrene : 1, 4 isoprene : 3, 4 isoprene
=A−4×C
9
5:C
9:E:D
2
(1)
Polymers 2021,13, 235 6 of 13
where Arepresents the peak area of nine protons on the styrenic aromatic ring (integral area
under Peak A), including five protons of styrene and four protons of tert-butyl styrene; C
expresses the nine protons of the tert-butyl group (integral area under Peak C); Daddresses
the single unsaturated proton of 1,4-isoprene (integral area under Peak D); Eindicates
two unsaturated protons of 3,4-isoprene (integral area under Peak E). The structural
characteristics of the different SIS copolymers are summarized in Table 1. It is noted that
the experimental styrene/tert-butyl styrene weight ratio increased with the amount of
styrene added. However, all styrene/tert-butyl styrene weight ratios were lower than
their theoretical counterparts, likely resulting from higher tert-butyl styrene reactivity than
styrene. All experimental styrenic block percentages (mole %) were similar to each other
and were higher than the theoretical counterparts.
Polymers 2021, 13, x FOR PEER REVIEW 6 of 13
addresses the single unsaturated proton of 1,4-isoprene (integral area under Peak D); E
indicates two unsaturated protons of 3,4-isoprene (integral area under Peak E). The struc-
tural characteristics of the different SIS copolymers are summarized in Table 1. It is noted
that the experimental styrene/tert-butyl styrene weight ratio increased with the amount of
styrene added. However, all styrene/tert-butyl styrene weight ratios were lower than their
theoretical counterparts, likely resulting from higher tert-butyl styrene reactivity than sty-
rene. All experimental styrenic block percentages (mole %) were similar to each other and
were higher than the theoretical counterparts.
After hydrogenation, the chemical shifts at 4.7 and 5.1 ppm, assigned to the vinyl
protons of the 3,4-isoprene and 1,4-isoprene units, respectively, disappeared (Figure 2).
Thus, we could confirm that the double bonds in the soft segments were fully saturated.
Figure 1. 1H-NMR spectra of styrene–isoprene–styrene (SIS) triblock copolymers of different feed
ratios.
Since the sulfonated block copolymers were insoluble in standard deuterium sol-
vents, the NMR analyses for these specimens were not executed. The degree of sulfonation
(DS, the ratio of the number of styrene units attached with the sulfonated group compared
to the total number of styrene unit), was calculated using Equation (2)
(2)
where V represents the volume of methanolic KOH titrant consumed, and C denotes the
molar concentration of KOH solution. m addresses the weight of styrene in the block co-
polymer, where M1 and M2 represent the molecular weight of tert-butyl styrene and sty-
rene.
Figure 1. 1
H-NMR spectra of styrene–isoprene–styrene (SIS) triblock copolymers of different feed ratios.
After hydrogenation, the chemical shifts at 4.7 and 5.1 ppm, assigned to the vinyl
protons of the 3,4-isoprene and 1,4-isoprene units, respectively, disappeared (Figure 2).
Thus, we could confirm that the double bonds in the soft segments were fully saturated.
Polymers 2021,13, 235 7 of 13
Polymers 2021, 13, x FOR PEER REVIEW 7 of 13
Table 1. Composition of different SIS copolymers.
S5T5
S6T4
S7T3
S8T2
S9T1
Styrene feeding weight (g)
1.0
1.2
1.4
1.6
1.8
tert-butyl styrene (tbs) feeding weight (g)
1.0
0.8
0.6
0.4
0.2
Isoprene feeding weight (g)
5.0
5.0
5.0
5.0
5.0
Styrene/tbs (experimental weight ratio) a
0.67
0.79
0.91
1.05
1.16
Styrenic block (theoretical mole %)
0.18
0.18
0.19
0.20
0.20
Styrenic block (experimental mole %) a
0.39
0.36
0.37
0.37
0.34
1,4 isoprene/3,4 isoprene (molar ratio) a
15.45
14.02
13.37
14.24
15.05
a Calculated from 1H NMR.
Figure 2. 1H-NMR spectra of different SIS copolymers after hydrogenation.
The DS for sulfonated samples sS5T5, sS6T4, sS7T3, sS8T2, and sS9T1 were 2.82 ±
0.29%, 1.68 ± 0.11%, 2.59 ± 0.28%, 0.78 ± 0.14%, and 1.10 ± 0.32%, respectively. Statistical
analyses revealed that the order of DS was as follows: S5T5–sS7T3 > sS6T4 > sS9T1–sS8T2.
The reason behind the difference in the degree of sulfonation might be linked to the dif-
ferent ratios of styrene and 4-tert-butyl styrene. In the S5T5 sample, the styrene units in
the hard segment could be well dispersed, and the sulfonic functional groups could be
introduced without high hindrance. Similarly, the higher amount of 4-tert-butyl styrene
that could be sulfonated (Scheme 1) would also lead to a higher DS noted. Hence, the
difference in the hard segment morphology and the amount of 4-tert-butyl styrene avail-
able for sulfonation could synergistically affect the DS noted.
The sulfonation of the polystyrene block was further confirmed by ATR-FTIR analy-
sis (Figure 3). A doublet positioned at 1200 cm–1 and a band at 1030 cm–1, corresponding
to the antisymmetric and symmetric stretching vibrations of SO3-, respectively, [31] were
noted, implicating the success of sulfonating the hydrogenated specimens.
Figure 2. 1H-NMR spectra of different SIS copolymers after hydrogenation.
Since the sulfonated block copolymers were insoluble in standard deuterium solvents,
the NMR analyses for these specimens were not executed. The degree of sulfonation (DS,
the ratio of the number of styrene units attached with the sulfonated group compared to
the total number of styrene unit), was calculated using Equation (2)
DS (mole %)=V×C
V×C+m−V×C×M1
M2
×100% (2)
where Vrepresents the volume of methanolic KOH titrant consumed, and Cdenotes the molar
concentration of KOH solution. maddresses the weight of styrene in the block copolymer,
where M1and M2represent the molecular weight of tert-butyl styrene and styrene.
The DS for sulfonated samples sS5T5, sS6T4, sS7T3, sS8T2, and sS9T1 were
2.82 ±0.29%
,
1.68
±
0.11%, 2.59
±
0.28%, 0.78
±
0.14%, and 1.10
±
0.32%, respectively. Statistical analyses
revealed that the order of DS was as follows: S5T5–sS7T3 > sS6T4 > sS9T1–sS8T2. The
reason behind the difference in the degree of sulfonation might be linked to the different
ratios of styrene and 4-tert-butyl styrene. In the S5T5 sample, the styrene units in the hard
segment could be well dispersed, and the sulfonic functional groups could be introduced
without high hindrance. Similarly, the higher amount of 4-tert-butyl styrene that could be
sulfonated (Scheme 1) would also lead to a higher DS noted. Hence, the difference in the hard
segment morphology and the amount of 4-tert-butyl styrene available for sulfonation could
synergistically affect the DS noted.
The sulfonation of the polystyrene block was further confirmed by ATR-FTIR analysis
(Figure 3). A doublet positioned at 1200 cm
−1
and a band at 1030 cm
−1
, corresponding to
the antisymmetric and symmetric stretching vibrations of SO
3−
, respectively, [
31
] were
noted, implicating the success of sulfonating the hydrogenated specimens.
Polymers 2021,13, 235 8 of 13
Polymers 2021, 13, x FOR PEER REVIEW 8 of 13
Figure 3. ATR-FTIR spectra of the hydrogenated S5T5 and sulfonated hydrogenated S5T5 (sS5T5).
The contact angle of hydrogenated and sulfonated hydrogenated SIS membranes
with different degrees of sulfonation is illustrated in Figure 4. As shown in Figure 4, the
hydrogenated SIS membrane showed a high water contact angle of about 106, which is
close to the values reported by Patio et al. [28]. The water contact angle of the sulfonated
membranes decreased progressively by increasing the degree of sulfonation, owing to the
incorporation of hydrophilic sulfonic groups. Interestingly, for the sS5T5 membrane with
the highest degree of sulfonation, the membrane was falling apart after contact with wa-
ter, and, hence, it was not used for subsequent analyses needed for water/PBS contact for
a while.
Figure 4. Contact angle results of the hydrogenated and sulfonated hydrogenated SIS samples.
Water uptake was determined by immersing the sample membranes into the deion-
ized water at room temperature for 12 h, removing any excess water on the surface, and
finally weighing them on a microbalance. The water uptake was:
𝑊(%)=𝑊𝑤 −𝑊𝑑
𝑊𝑑 ×100
Figure 3. ATR-FTIR spectra of the hydrogenated S5T5 and sulfonated hydrogenated S5T5 (sS5T5).
The contact angle of hydrogenated and sulfonated hydrogenated SIS membranes with
different degrees of sulfonation is illustrated in Figure 4. As shown in Figure 4, the hydro-
genated SIS membrane showed a high water contact angle of about 106
◦
, which is close to the
values reported by Patiño et al. [
28
]. The water contact angle of the sulfonated membranes
decreased progressively by increasing the degree of sulfonation, owing to the incorporation of
hydrophilic sulfonic groups. Interestingly, for the sS5T5 membrane with the highest degree of
sulfonation, the membrane was falling apart after contact with water, and, hence, it was not
used for subsequent analyses needed for water/PBS contact for a while.
Polymers 2021, 13, x FOR PEER REVIEW 8 of 13
Figure 3. ATR-FTIR spectra of the hydrogenated S5T5 and sulfonated hydrogenated S5T5 (sS5T5).
The contact angle of hydrogenated and sulfonated hydrogenated SIS membranes
with different degrees of sulfonation is illustrated in Figure 4. As shown in Figure 4, the
hydrogenated SIS membrane showed a high water contact angle of about 106, which is
close to the values reported by Patio et al. [28]. The water contact angle of the sulfonated
membranes decreased progressively by increasing the degree of sulfonation, owing to the
incorporation of hydrophilic sulfonic groups. Interestingly, for the sS5T5 membrane with
the highest degree of sulfonation, the membrane was falling apart after contact with wa-
ter, and, hence, it was not used for subsequent analyses needed for water/PBS contact for
a while.
Figure 4. Contact angle results of the hydrogenated and sulfonated hydrogenated SIS samples.
Water uptake was determined by immersing the sample membranes into the deion-
ized water at room temperature for 12 h, removing any excess water on the surface, and
finally weighing them on a microbalance. The water uptake was:
𝑊(%)=𝑊𝑤 −𝑊𝑑
𝑊𝑑 ×100
Figure 4. Contact angle results of the hydrogenated and sulfonated hydrogenated SIS samples.
Water uptake was determined by immersing the sample membranes into the deionized
water at room temperature for 12 h, removing any excess water on the surface, and finally
weighing them on a microbalance. The water uptake was:
W(%)=Ww −W d
Wd ×100
Polymers 2021,13, 235 9 of 13
where Wd and Ww are the mass of the dry sample and the mass of the water-swollen
sample, respectively. It was noted that the water uptake values for the sulfonated samples
of sS6T4, sS7T3, sS8T2, and sS9T1 were 30.19
±
2.27%, 40.63
±
1.79%, 18.91
±
0.21%, and
8.47
±
0.65%, respectively. These values correlated with the degree of sulfonation of the
tested specimens.
The surface charge density could have played an important role when the material
was in contact with the physiological environment. The streaming potential of the nonsul-
fonated hydrogenated SIS membranes and sulfonated membranes is shown in Figure 5.
With the incorporation of anionic sulfonic functional groups, the membrane surface became
more negatively charged compared to the nonsulfonated groups, except S8T2. However,
there were no significant differences among the sulfonated samples, except sS8T2, on which
the streaming potential was more statistically positive.
Polymers 2021, 13, x FOR PEER REVIEW 9 of 13
where Wd and Ww are the mass of the dry sample and the mass of the water-swollen
sample, respectively. It was noted that the water uptake values for the sulfonated samples
of sS6T4, sS7T3, sS8T2, and sS9T1 were 30.19 ± 2.27%, 40.63 ± 1.79%, 18.91 ± 0.21%, and
8.47 ± 0.65%, respectively. These values correlated with the degree of sulfonation of the
tested specimens.
The surface charge density could have played an important role when the material
was in contact with the physiological environment. The streaming potential of the non-
sulfonated hydrogenated SIS membranes and sulfonated membranes is shown in Figure
5. With the incorporation of anionic sulfonic functional groups, the membrane surface
became more negatively charged compared to the nonsulfonated groups, except S8T2.
However, there were no significant differences among the sulfonated samples, except
sS8T2, on which the streaming potential was more statistically positive.
Figure 5. Streaming potential of the hydrogenated and sulfonated hydrogenated SIS samples.
3.2. Cytotoxicity Test
Cell viability testing was required for the material intended for biomedical applica-
tions. Following the guidance of ISO 10993-5 and ISO 10993-12, which focused on the cell
viability testing on the extracts of the sulfonated copolymers (Figure 6A) and direct con-
tact with the copolymers (Figure 6B). The difference in cell viability values between these
two assays could be attributed to the difference in the film thickness used: 8 mm for the
extract assay and 1 mm for the direct-contact experiment. Nevertheless, the film thickness
of the sulfonated copolymers will be within this tested range for future applications. The
results have shown all sulfonated materials presented over 70% cell viability value, either
by testing the extract or by direct contact, required by the ISO standard for nontoxic ma-
terials.
Figure 5.
Streaming potential of the hydrogenated and sulfonated hydrogenated SIS samples.
3.2. Cytotoxicity Test
Cell viability testing was required for the material intended for biomedical applica-
tions. Following the guidance of ISO 10993-5 and ISO 10993-12, which focused on the cell
viability testing on the extracts of the sulfonated copolymers (Figure 6A) and direct contact
with the copolymers (Figure 6B). The difference in cell viability values between these two
assays could be attributed to the difference in the film thickness used: 8 mm for the extract
assay and 1 mm for the direct-contact experiment. Nevertheless, the film thickness of the
sulfonated copolymers will be within this tested range for future applications. The results
have shown all sulfonated materials presented over 70% cell viability value, either by
testing the extract or by direct contact, required by the ISO standard for nontoxic materials.
3.3. In Vitro Platelet Adhesion Testing
The adhesion and activation of platelets on material surfaces could initiate throm-
boembolic complications. Therefore, there is a need to conduct
in vitro
platelet adhesion
tests to evaluate the biocompatibility of our samples. Figures 7and 8show the results
of the
in vitro
platelet adhesion test on the hydrogenated and sulfonated hydrogenated
copolymer surfaces. All sulfonated samples demonstrated a higher amount of platelet
adhesion density than the unsulfonated counterparts. Nevertheless, most of the adhered
platelets remained unactivated or only slightly activated (Figure 9).
Polymers 2021,13, 235 10 of 13
Polymers 2021, 13, x FOR PEER REVIEW 11 of 15
(a)
(b)
Figure 6. Cytotoxicity assay on different sulfonated copolymers by (a) the extracts and (b) direct contact.
3.3. In Vitro Platelet Adhesion Testing
The adhesion and activation of platelets on material surfaces could initiate thrombo-
embolic complications. Therefore, there is a need to conduct in vitro platelet adhesion tests
to evaluate the biocompatibility of our samples. Figures 7 and 8 show the results of the in
vitro platelet adhesion test on the hydrogenated and sulfonated hydrogenated copolymer
surfaces. All sulfonated samples demonstrated a higher amount of platelet adhesion den-
sity than the unsulfonated counterparts. Nevertheless, most of the adhered platelets re-
mained unactivated or only slightly activated (Figure 9).
Such an increase in platelet adhesion density but with an unactivated morphology
was also noted on the sulfonate-incorporated polyether polyurethane [32] as well as our
earlier study on the N,O-sulfated chitosan [33]. This is likely due to the surface-negative
charge at the physiological incubation condition, which changed the adsorbed protein
composition and conformation, resulting in increased platelet adhesion while not being
Figure 6. Cytotoxicity assay on different sulfonated copolymers by (a) the extracts and (b) direct contact.
Polymers 2021, 13, x FOR PEER REVIEW 11 of 13
Figure 7. SEM micrographs (3000×) for the platelets adhered onto the hydrogenated (a) S6T4, (c) S7T3, (e) S8T2, (g) S9T1
and sulfonated hydrogenated (b) sS6T4, (d) sS7T3, (f) sS8T2, and (h) sS9T1 (scale bar: 1 µm).
Figure 8. Platelet adhesion density on the hydrogenated and sulfonated block copolymers.
Figure 9. Number of activated (i.e., not in a round shape) adherent platelets per 1000 µm2 on the
hydrogenated and sulfonated block copolymers.
Figure 7.
SEM micrographs (3000
×
) for the platelets adhered onto the hydrogenated (
a
) S6T4, (
c
) S7T3, (
e
) S8T2, (
g
) S9T1
and sulfonated hydrogenated (b) sS6T4, (d) sS7T3, (f) sS8T2, and (h) sS9T1 (scale bar: 1 µm).
Polymers 2021,13, 235 11 of 13
Polymers 2021, 13, x FOR PEER REVIEW 11 of 13
Figure 7. SEM micrographs (3000×) for the platelets adhered onto the hydrogenated (a) S6T4, (c) S7T3, (e) S8T2, (g) S9T1
and sulfonated hydrogenated (b) sS6T4, (d) sS7T3, (f) sS8T2, and (h) sS9T1 (scale bar: 1 µm).
Figure 8. Platelet adhesion density on the hydrogenated and sulfonated block copolymers.
Figure 9. Number of activated (i.e., not in a round shape) adherent platelets per 1000 µm2 on the
hydrogenated and sulfonated block copolymers.
Figure 8. Platelet adhesion density on the hydrogenated and sulfonated block copolymers.
Polymers 2021, 13, x FOR PEER REVIEW 11 of 13
Figure 7. SEM micrographs (3000×) for the platelets adhered onto the hydrogenated (a) S6T4, (c) S7T3, (e) S8T2, (g) S9T1
and sulfonated hydrogenated (b) sS6T4, (d) sS7T3, (f) sS8T2, and (h) sS9T1 (scale bar: 1 µm).
Figure 8. Platelet adhesion density on the hydrogenated and sulfonated block copolymers.
Figure 9. Number of activated (i.e., not in a round shape) adherent platelets per 1000 µm2 on the
hydrogenated and sulfonated block copolymers.
Figure 9.
Number of activated (i.e., not in a round shape) adherent platelets per 1000
µ
m
2
on the hydrogenated and sulfonated block copolymers.
Such an increase in platelet adhesion density but with an unactivated morphology was
also noted on the sulfonate-incorporated polyether polyurethane [
32
] as well as our earlier
study on the N,O-sulfated chitosan [
33
]. This is likely due to the surface-negative charge at
the physiological incubation condition, which changed the adsorbed protein composition
and conformation, resulting in increased platelet adhesion while not being activated. It was
of note that the sulfonated S8T2 presented the lowest platelet adhesion density and had
the least negative streaming potential (Figure 5). This further substantiated the importance
of surface charge when coming into contact with blood/platelets.
4. Conclusions
By controlling the weight ratio of tert-butyl styrene in the hydrogenated SIS block copoly-
mers, sulfonated copolymeric films were demonstrated to have hydrophilic swollen charac-
teristics without losing their physical intactness. Biological assays showed these sulfonated
samples are noncytotoxic. Although the platelet adhesion assay indicated these sulfonated
films could not effectively decrease the platelet adhesion density, the adhered platelets were
still not activated and, hence, the least thrombogenic-prone when in contact with the whole
blood. Further studies will be required for optimizing the physical performance of sulfonated
hydrogenated SIS as a potential alternative for wound-dressing applications.
Polymers 2021,13, 235 12 of 13
Author Contributions:
Conceptualization, B.-H.T. and J.-C.L.; Data Curation, B.-H.T. and T.-A.L.;
Formal Analysis, B.-H.T., T.-A.L. and J.-C.L.; Funding Acquisition, C.-H.C. and J.-C.L.; Investigation,
B.-H.T. and T.-A.L.; Methodology, B.-H.T. and T.-A.L.; Project Administration, J.-C.L.; Resources,
C.-H.C. and J.-C.L.; Validation, B.-H.T. and T.-A.L.; Writing—Original Draft Preparation, B.-H.T. and
J.-C.L.; Writing—Review & Editing, B.-H.T., T.-A.L., C.-H.C. and J.-C.L. All authors have read and
agreed to the published version of the manuscript.
Funding:
The authors would like to acknowledge the financial support from the Ministry of Science
and Technology, Taiwan under Grant MOST106-2221-E-006-203-MY3, MOST109-2622-E-006-012-CC3,
MOST109-2221-E-006-105-MY3, MOST107-2314-B-182-043, MOST108-2314-B-182-036, and MOST109-
2314-B-182-039. The financial support from the Chang Gung Memorial Hospital under Grant CMRPG
3C1583, CMRPG 3D1283, CMRPG 3H0181, CMRPG 3H0182, and CMRPG 3H0183 is also acknowl-
edged.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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