Differences in Zwitterionic Sulfobetaine and Carboxybetaine Dextran-Based Hydrogels

Article abstract of DOI:10.1021/acs.langmuir.8b01869

Zwitterionic sulfobetaine (SB) and carboxybetaine (CB) have been extensively investigated for their noticeable antifouling properties. Both SB and CB have cationic and anionic groups in the molecule, but they differ in negatively charged groups. Molecular simulations have been conducted to investigate the different properties induced by structure changes. However, few studies have focused on the differences between SB and CB materials, especially zwitterionic polysaccharides. Two zwitterionic sulfobetaine and carboxybetaine dextran hydrogels were designed and used as models to compare their properties. Results showed that the equilibrium swelling ratios of the SB-DEX hydrogels were much higher than CB-DEX ones, and larger interior pores were observed in the SB-DEX hydrogels due to their higher hydrophilicity. The rheological storage modulus of the SB-DEX hydrogels was lower than that of CB-DEX ones as a result of higher water content of SB-DEX. These results were consistent with molecular modeling. Additionally, both CB-DEX and SB-DEX had remarkable biocompatibilities, and the in vitro release studies showed that the SB-DEX and CB-DEX hydrogels released DOX in a sustained manner under acidic condition (pH 5.0), indicating their promise as an effective drug-delivery system.

Full text of DOI:10.1021/acs.langmuir.8b01869

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Differences in zwitterionic sulfobetaine and
carboxybetaine dextran based hydrogels
Xiaofeng Chen, Xia Qiu, Minghong Hou, Xiaotian Wu,
Yahao Dong, Yansong Ma, Li-Jun Yang, and Yuping Wei
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01869 • Publication Date (Web): 24 Aug 2018
Downloaded from http://pubs.acs.org on August 28, 2018
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Figure 1. Synthesis of the zwitterionic sulfobetaine (SB).
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Figure 2. Preparation of the SB-DEX hydrogel.
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Figure 3. 1H-NMR spectra of (A) dextran, (B) CB-DEX, (C) SB-DEX, (D) SB.
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Figure 4. FT-IR spectra of (A) SB-DEX hydrogel, (B) CB-DEX hydrogel, (C) dextran, (D) SB-DEX.
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Figure 5.The swelling ratios of the SB-DEX hydrogels and CB-DEX hydrogels.
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Figure 6. The SEM images of the SB-DEX and CB-DEX hydrogels.
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Figure 7.The microcalorimetric titration curves for (A) dextran and (B) SB-DEX.
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Figure 8. The storage modulus of the CB-G0.20 and SB-G0.20 hydrogels.
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Figure 9. The cumulative DOX release from drug loaded SB and CB hydrogels in different pH conditions. (A)
SB-G0.20 in pH=5.0, (B) SB-G0.20 in pH=7.4, (C) CB-G0.20 in pH=5.0, (D) CB-G0.20 in pH=7.4.
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Figure 10. The cell viability of Dextran, SB-G0.20 and CB-G0.20 hydrogels.
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Figure 11. The cell viability of free DOX and DOX loaded hydrogels (SB-G0.20/DOX and CB-G0.20/DOX).
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TOC graphic
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Differences in zwitterionic sulfobetaine and carboxybetaine dextran
based hydrogels
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Xiaofeng Chena, Xia Qiua, Minghong Houa, Xiaotian Wua, Yahao Donga, Yansong
Maa, Lijun Yang*,b, Yuping Wei*,a,c
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aDepartment of Chemistry, School of Science, Tianjin University, Tianjin 300354, PR
China
bTianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,
Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking
Union Medical College, Tianjin 300192, PR China
cCollaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, PR China
*Corresponding Author. E-mail: yanglijunly@126.com (L. Yang), ypwei@tju.edu.cn
(Y. Wei). Fax: +86-22-27403475.
Abstract
Zwitterionic sulfobetaine (SB) and carboxybetaine (CB) have been extensively
investigated for their noticeable antifouling properties. Both SB and CB have cationic
and anionic groups in the molecule but they differ in negatively charged groups.
Molecular simulations have been conducted to investigate the different properties
induced by structure changes. However, few studies have focused on the differences
between SB and CB materials especially zwitterionic polysaccharides. In this paper,
two zwitterionic sulfobetaine and carboxybetaine dextran hydrogels were designed
and used as models to compare their properties. Results showed that the equilibrium
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swelling ratios of the SB-DEX hydrogels were much higher than CB-DEX ones and
larger interior pores were observed in the SB-DEX hydrogels due to their higher
hydrophilicity. Rheological storage modulus of the SB-DEX hydrogels was lower
than CB-DEX ones as a result of higher water content of SB-DEX. These results were
consistent with molecular modeling. Additionally, both of CB-DEX and SB-DEX had
remarkable biocompatibilities. And the in vitro release studies showed the SB-DEX
and CB-DEX hydrogels released DOX in a sustained manner in acidic condition (pH
5.0), indicating the promise as effective drug delivery system.
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Introduction
Hydrogels are soft and moisty materials consisting of three-dimensional network
cross-linked via covalent or non-covalent bonds, swelling rapidly to equilibrium
without dissolution.1,2 Hydrogels have been extensively investigated in biomedical
applications such as biosensing,3 tissue engineering,4,5 wound-dressing6 and drug
delivery,7-9 due to their high water content, excellent biocompatibility and great
potential for drug loading. Despite their advantages, however, most hydrogel implants
suffer from the non-specific protein adsorption and immunological response,10-12
triggering bacterial adhesion and chronic inflammation.11,12 Biomaterial-associated
fouling accounts for ~45% of the nosocomial infections.13 Therefore, it’s critical to
develop new materials with desired antifouling property as well as excellent
biocompatibility.
Recently, zwitterionic polymers have been found to be a new generation of
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antifouling materials. Zwitterionic materials have both cationic and anionic groups in
one molecule while neutrally charged as a whole. The antifouling ability of
zwitterionic polymers was attributed to the static induced hydration layer.12,14 The
unique structure facilitates their increasing applications in biological fields.15,16 For
example, zwitterionic polymer brushes have been demonstrated as promising stealth
coatings due to the excellent antifouling performance in human blood.12 Previously,
most studies focused on the zwitterionic co-polymers based on polymethacrylate or
polyacrylamide backbone.17-19 However, more biocompatible materials are required
for sustainable development.
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Polysaccharides have attracted increasing research interests because of their
non-toxicity, protein-resistant feature, tunable structure, and abundance in nature.8,20
Particularly, zwitterionic polysaccharides were able to activate CD4+ T cells and thus
prevent from abscess formation.21,22 Recently, a series of zwitterionic polysaccharides
have been developed. For example, zwitterionic SB starch based hydrogels were
fabricated for cell encapsulation and showed high resistance to nonspecific protein
and cell adhesion.23 Cyclodextrin hydrogels with zwitterionic SB guest were reported
to retain the beneficial nature of native cyclodextrin.24 Among these, dextran and its
derivatives have been extensively exploited in biomedical applications owing to their
good water solubility and antifouling properties. For example, zwitterionic CB was
introduced into dextran backbone to endow the hydrogel platform with excellent
antifouling property and minimized cell attachment.11 All these studies indicated that
both CB and SB modified polysaccharides perform well in resisting protein
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adsorption although they differ in negatively charged headgroups (COO- for CB and
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-
SO3 for SB). Further, the difference of negative charges may cause varying hydration
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properties as predicted in Jiang group’s works.25-28 Therefore, to distinguish SB and
CB functionalized polysaccharides on the molecular level is an very important issue
needed to be solved.
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In this paper, two zwitterionic SB and CB dextran hydrogels were designed and
synthesized as models. In order to figure out if the two hydrogels will show different
properties as predicted in previous simulations, various properties including
equilibrium swelling ratio, surface zeta potential, interior morphology and rheological
properties were investigated. Furthermore, the potential for drug delivery was also
studied using doxorubicin (DOX) as a model drug. Additionally, the cytotoxicity
experiments were performed.
Experimental section
Materials
Dextran (Mn=40000) was purchased from Sinopharm Chemical Reagent Co.,
Ltd. (99%) and used as received. Dimethylsulfoxide (DMSO) was obtained from
Tianjin Fuchen Chemical Industry Co., Ltd., distilled under reduced pressure and
stored over molecular sieves. Di-tert-butyldicarbonate and propane sultone were
purchased from Tianjin Heowns Biochem Technologies Co., Ltd. and used as
received. N,N  -dimethylethylenediamine and N,N -carbonyldiimidazole (CDI) were
obtained
from
Shanghai
Dibai
Biological
Technology
Co.,
Ltd.
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4-(N,N-dimethylamino)-pyridine (DMAP) was obtained from Beijing HWRK Chem
Co., Ltd. (99%) and used as received. Chloroform, methanol and ethyl acetate were
purchased from Tianjin Yuanli Chemical Industry Co., Ltd. and used without further
purification. Fibrinogen was provided by Shanghai Yuanye Biological Technology
Co., Ltd. and stored in 4 °C environment before used. Doxorubicin hydrochloride
(DOX•HCl) was obtained from Dalian Meilun Biology Technology Co., Ltd. (98%)
and stored in 4 °C environment. Dialysis tubes (MWCO 7000) were purchased from
Sigma.
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Synthesis of SB
Zwitterionic sulfobetaine (SB) was synthesized as shown in Figure 1.
N-Boc-dimethylethylenediamine (a). Di-tert-butyldicarbonate (9.17 g, 42 mmol) was
added to the solution of N,N-dimethylethylenediamine (3.61 g, 40 mmol) in 50 mL
chloroform at 0 °C dropwise and then the mixture was stirred overnight at room
temperature. The solvent was removed by evaporation under vacuum. Thereafter, the
same volume of water and ethyl acetate was added for extraction. The oil-like product
was obtained (6.07 g, yield 80.68%) by concentrating the extracts under reduced
pressure. The 1H-NMR spectrum is shown in Figure S1.
3-((3-((tert-Butoxycarbonyl)amino)ethyl)-dimethylammonio)propane-1-sulfonate
(Boc-SB, b). Propane sultone (5.91 g, 48.41 mmol) was dissolved in
dimethylformamide
and
added
dropwise
to
the
solution
of
N-Boc-dimethylethylenediamine (6.07 g, 32.27 mmol), and this mixture was kept
stirring for 2 days at room temperature. The solvent was evaporated and the
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precipitate was rinsed with ethyl acetate for three times and collected by filtration,
then it was dried under vacuum for 12 h. The resulting white solid was obtained (7.05
g, yield 70.50%). The 1H-NMR spectrum is shown in Figure S2.
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3-((2-aminoethyl)-dimethylammonio)propane-1-sulfonate (sulfobetaine, c). 10%
HCl in methanol was utilized in the removal of the Boc groups. The solid of b (7.05 g,
22.75 mmol) was dissolved in 20 ml methanol, and excess amount of HCl/MeOH was
dropped into the solution at 0 °C, then the mixture was warmed to room temperature
and left stirring for overnight. The final product was obtained as white powders (4.72
g, yield 98.86%) by centrifugation and drying.
Figure 1. Synthesis of the zwitterionic sulfobetaine (SB).
Synthesis of SB-DEX
The sulfobetaine (SB) was covalently attached onto the dextran backbone via the
amidation of the amine and hydroxyl groups using CDI as the activation agent.
Dextran (0.16 g, 1 mmol) was dissolved in 15 ml dried DMSO. After dissolving
DMAP (0.012 g), CDI (0.48 g, 3 mmol) was added to activate the hydroxyl group of
dextran. The reaction mixture was stirred at 50 °C for 1 h under nitrogen atmosphere.
Then SB (0.21 g, 1 mmol) was added, and the solution was left stirring at 50 °C for 48
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h under nitrogen atmosphere. The polymer solution was dialyzed against distilled
water and then lyophilized to give the sulfobetaine conjugated dextran (SB-DEX) as
white powders (0.19 g, yield 47.38%).
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Synthesis of CB-DEX
CB-DEX was prepared as described in our previous work.29 Dextran (1.50 g,
9.23 mmol) was dissolved in 50 ml NaOH solution (0.08 g/ml), then 5 ml of
epichlorohydrin (63.77 mmol) was added and the solution was stirred for 16 h at
30 °C, and 2 g NaOH was dissolved in the mixture before the addition of
N,N-dimethylglycine (5.00 g, 48.49 mmol), and the solution was then allowed to stir
for 3 days at 55 °C. Afterwards, the resulting mixture was dialyzed against distilled
water for 2 days and then lyophilized. The CB-DEX was obtained as white powders
(1.95 g, yield 33.68%).
Preparation of SB and CB dextran based hydrogels
Hydrogels were prepared following the previously reported method.2 As shown
in Figure 2, SB-DEX (or CB-DEX) was dissolved in 2 M NaOH aqueous solution,
and then different amounts of crosslinking agent was added to the polymer solution,
and the mixture was kept at 50 °C for 1 h to give three dimensional reticulated
structures. The molar ratios of MBA/SB-DEX (or CB-DEX) were 0.15, 0.20, 0.25,
respectively. The detailed dosage of regents used in hydrogel preparation was shown
in Table 1. The formation of hydrogels was characterized by the vial-tilting method.
The resulting hydrogels were immersed in distilled water for 7 days to remove the
unreacted chemicals and then lyophilized.
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Table1. The dosage of regents used in hydrogel preparation
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Mass of zwitterionics (g)
Hydrogel
samples
Mass of
Molar ratio of
Volume of
MBA (g)
MBA/zwitterionics NaOH (mL)
SB-DEX
CB-DEX
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SB-G0.15
SB-G0.20
SB-G0.25
CB-G0.15
CB-G0.20
CB-G0.25
0.282
0.282
0.282
0
0
0.016
0.022
0.028
0.016
0.022
0.028
0.15
0.20
0.25
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Figure 2. Preparation of the SB-DEX hydrogel.
1H-NMR and FT-IR characterization
1H-NMR spectra of the synthesized SB, SB-DEX and CB-DEX were measured
by a Bruker Avance III 400 MHz spectrometer in D2O using TMS as the internal
standard. The samples for FT-IR characterization were prepared in KBr pellets. The
FT-IR tests were performed using a Nicolet 5DXC FTIR spectrometer with a scanning
range of 4000-400 cm-1 and a resolution of 4 cm-1.
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Swelling characterization
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The swelling behavior of the hydrogels were measured gravimetrically2,23 as the
following procedures. The prepared hydrogels were immersed in distilled water for 7
days to remove the uncrosslinked polymers, and then the samples were taken out,
followed by removing excess water on the surface using filter papers, the swollen
hydrogels were weighed immediately and the equilibrium swelling ratio was
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calculated based on the equilibrium weight ( W
e
) and the freeze-dried weight ( W ).
0
All tests were performed in triplicate.
Equilibrium Swelling Ratio =

We
 W
0

W
0
(1)
Scanning electron microscope (SEM) characterization
The morphology of the hydrogels was investigated using a scanning electron
microscope (S-4800, Hitachi, Japan). The swollen hydrogels were lyophilized at
−40 °C for 48 h. Then the freeze-dried samples were putter-coated with a thin layer of
gold to enhance conductivity for the investigation of internal morphology.
Zeta potential measurements
The hydrogels prepared were crushed and soaked in distilled water for overnight.
The zeta potentials of the hydrogel samples were then measured using the zeta/nano
particle analyzer (Micromeritics, Co., China) with 20 scans for each sample. The
average zeta potentials were directly calculated and recorded by the instrument.
Isothermal titration calorimetry (ITC)
The ITC200 microcalorimeter was utilized to evaluate the resistance of the
zwitterionic SB-DEX against protein. Fibrinogen was chosen as a representative
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hydrophobic protein. Dextran and SB-DEX were dissolved in PBS buffer solution
(pH = 7.4, 0.01 M) with the concentration of 1.82 M. Titration was performed by
injecting the fibrinogen solution with the concentration of 7.35  M into a 280  L
polymer solution. For each time, 2  L fibrinogen solution was injected, and 120
second pauses between injections were allowed for the solution to reach equilibrium.
The generated calories for the interactions between the polymer and protein solutions
were recorded by the instrument.
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Dynamic rheology
Rheological experiments were carried out using a modular advanced rheometer
system, (HAAKE MARS, Germany). The storage modulus ( G' ) and loss modulus
(G'' ) were determined by time-sweep mode at a constant oscillator frequency (0.5 Hz)
and strain (0.1%) using a 35 mm diameter parallel plate geometry and a 0.5 mm gap.
All measurements were performed at 50 °C.
DOX loading and in vitro release
For DOX loading, 6.5 mg of the dried SB-G0.20 (or CB-G0.20) hydrogels were
immersed in 2.0 mL DOX aqueous solution (0.5 mg/mL) in a tube, and the mixture
was spinning at 100 rpm for 48 h at 37 °C in dark. Then the hydrogels were taken out
and washed with distilled water for three times to remove the unloaded DOX on the
surface. The supernatant was collected for UV-Vis spectral analysis (481 nm) of the
unloaded DOX.20 The drug loading efficiency in the hydrogels was calculated based
on the decrease of the original amount of DOX before drug loading.30 The standard
curve of DOX is shown in Figure S5. The encapsulation efficiency ( EE ) and loading
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capacity (LC ) were calculated as follows:
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DOX weight in the hydrogels
Feed DOX weight
DOX weight in the hydrogels
Total weight of hydrogels
EE (%) =
LC (%) =
× 100 %
× 100 %
(2)
(3)
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For the controlled release, DOX-loaded hydrogels (6.5 mg) were immersed in
PBS buffer (5.0 mL) with different pH, 5.0 and 7.4 respectively. The in vitro process
was carried out at 37 °C. At desired time intervals, 3.0 mL DOX contained buffer was
taken out to measure the DOX concentration by UV-Vis spectrophotometer (481 nm).
The equal volume of corresponding fresh PBS buffer was added to maintain the total
volume of the release buffer. The cumulative drug release percentage (RP) was
calculated as follows:
n1
Va Ci  VoCn

1
RP (%) =
× 100 %
(4)
M
Where M represents the weight of DOX in the hydrogels, and Va is the
withdrawn buffer volume per time ( Va = 3.0 mL), and Vo represents the whole
volume of the release buffer ( Vo = 5.0 mL), and Ci is the concentration of DOX in
the specific withdrawn buffer. All tests were performed in triplicate.
In vitro cytotoxicity assays
The cytocompatibility of the blank hydrogels was evaluated using NIH3T3 cells.
The hydrogels were sterilized by 75% ethanol before the cytotoxicity assays. The
NIH3T3cells suspension was added into a 96-well culture plate at a density of 8000
per well in 100 L of DMEM and incubated at 37 °C, 5% CO2 for 24 h. The original
culture medium was replaced by the same volume of fresh medium containing dextran,
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CB-G0.20 or SB-G0.20 hydrogels with different concentrations (0.05, 0.1, 0.2, 0.4,
0.6, 0.8 and 1 mg/mL). Cells without hydrogels treatment were used as the control.
After being incubated for 48 h, 20  L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) stock solution (5 mg/mL in PBS) was added to each well,
and the cells were incubated for another 4 h. Then, the medium was aspirated, and
150  L DMSO was added to dissolve the formed blue formazan crystals. The
absorbance were measured at 570 nm with a microplate reader (Varioskan Flash,
USA). The experiments were performed three times separately. The antiproliferative
activity of the DOX loaded hydrogels against Hela tumor cells was also examined by
MTT assays in a similar way. Hela cells were incubated with free DOX, DOX loaded
CB-G0.20 or SB-G0.20 hydrogels (CB-G0.20/DOX or SB-G0.20/DOX) with
different DOX concentrations (0.5, 1, 2.5, 5, 10, 20 and 50 g/mL) for 48 h. The cell
viability was normalized to the control group cultured with complete DMEM and
each sample was measured in triplicate.
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Results and discussion
1H-NMR and FT-IR characterization
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O
O
f
O
HO
HO
d
CB-DEX
SB-DEX
e
O
HO
HO
a
OH
b
c
OH
O
m
H3C
O
O
i
O
HO
HO
H3C
N+
N+
O
O
O
HO
O-
O
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g
O-
S
k
O
OH
CH3
NH
OH
O
O
h
l
j
CH3
i
O
D2O
i
g,h
j
l
k
SB
(D)
a
m
l
k
SB-DEX
CB-DEX
Dextran
(C)
(B)
(A)
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Chemical Shift (ppm)
Figure 3. 1H-NMR spectra of (A) dextran, (B) CB-DEX, (C) SB-DEX, (D) SB.
1
In Figure 3, the H-NMR spectra of natural dextran, CB-DEX, SB-DEX and SB
are presented. As shown in Figure 3(D), the remarkable peak centered at 3.16 ppm (i,
6H) is assigned to the methyl groups connected to the quaternary ammonium. The
peaks of propane sultone residues appear at 2.93 ppm (l, 2H), 2.19 ppm (k, 2H) and
3.66 ppm (j, 2H), and the peak at 3.50 ppm (g, h, 4H) is assigned to methylene
protons of N,N-dimethylethylenediamine. As indicated in Figure 3(C), the peaks at
4.88 ppm (a, 1H) and at the range of 4.0-3.5 ppm are assigned to dextran backbone,
and the new peaks observed at 3.08 ppm (i, 6H), 2.90 ppm (l, 2H) and 2.15 ppm (k,
2H) are assigned to the corresponding protons of SB, indicating the introduction of
1
SB onto the dextran backbone. The grafting ratio determined by H-NMR integration
is 16%, DS = Ii/6Ia. In addition to the original signals of natural dextran, the new peak
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at 3.19 ppm (m, 6H) in Figure 3(B) is attributed to the methyl groups adjacent to the
quaternary ammonium in CB-DEX. Based on the integration data from the anomeric
protons of dextran (a, 4.88 ppm) and the methyl groups of carboxybetaine (m, 3.19
ppm), the calculated grafting ratio is 15.5%, DS = Im/6Ia.
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(A)
SB-DEX hydrogel
(B)
CB-DEX hydrogel
Dextran
(C)
(D)
SB-DEX
1710
1545
1249
2936
3000
3407
3500
4000
2500
2000
1500
1000
500
wavenumber (cm-1)
Figure 4. FT-IR spectra of (A) SB-DEX hydrogel, (B) CB-DEX hydrogel, (C) dextran, (D)
SB-DEX.
The FT-IR spectrum also confirms the chemical structures of the zwitterionic
dextran hydrogels. As shown in Figure 4, the broad band ranging from 3000 to 3600
cm-1 is due to the -OH stretching vibration of the dextran backbone, and the band at
2936 cm-1 is assigned to -CH2 stretching vibration. Compared with unmodified
dextran, the peaks at 3407 cm-1 are weakened in Figure 4(D), indicating the
participation of the dextran hydroxyl groups. Besides, the remarkable absorption band
at 1710 cm-1 in the SB-DEX spectrum is attributed to C=O stretching vibration, and
the absorption at 1545 cm-1 is assigned to the in-plane bending vibration of N-H,
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suggesting the formation of the COONH carbamate. Furthermore, the appearance of
the absorption at 1249 cm-1 corresponding to the C-O bond of the carbamate group
also suggests the grafting of sulfobetaine onto the dextran backbone. The FT-IR
spectra of the SB-DEX hydrogel and CB-DEX hydrogel are similar in the weakened
signal at 3407 cm-1, substantiating the consumption of the hydroxyl groups during
hydrogel formation. Additionally, the absorptions of the SB-DEX hydrogel at 1710
cm-1 and 1545 cm-1 represent the characteristic bands of sulfobetaine.
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Swelling characterization.
Plots of the swelling ratios of the prepared SB-DEX and CB-DEX hydrogels
versus crosslinking degrees are shown in Figure 5. The swelling ratio values decrease
with increasing crosslinking degrees for both SB-DEX and CB-DEX hydrogels.
Interestingly, the equilibrium ratios of the SB-DEX hydrogels are much higher than
the CB-DEX system at the same crosslinking degree. The swelling ratio is 29.39 for
SB-G0.15 compared to 10.28 for CB-G0.15. The SB-G0.20 has the swelling value of
16.34 while for CB-G0.20 it is 9.45. The swelling ratio is decreased to 8.48 for
CB-G0.25 compared to 9.89 for SB-G0.25. The difference was probably because the
SB-DEX was more hydrophilic thus enhanced the affinity of hydrogels for water. As
described in previous work by the Jiang’s group,25 the volume of first coordination
shell of sulfobetaine is larger than that of carboxybetaine because of the extra oxygen
atoms in the negatively charged groups. The coordination numbers (N) of water
molecules in the first coordination shell showed that these two zwitterionic molecules
have similar N of the positively charged groups, however varying N of the negatively
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charged groups, indicating that sulfobetaine has more water molecules around it than
carboxybetaine. This is in accord with the remarkable difference of swelling ratios of
the SB-DEX and CB-DEX hydrogels.
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Figure 5.The swelling ratios of the SB-DEX hydrogels and CB-DEX hydrogels.
SEM characterization
The morphologies of the SB-DEX and CB-DEX hydrogels are shown in Figure 6.
All samples exhibit interconnected porous network structure. The pore size of
SB-DEX hydrogels was larger than the CB-DEX hydrogels for three crosslinking
degrees, due to the different hydrophilicity of sulfobetaine and carboxybetaine.
Sulfobetaine can absorb more water25 and thus the SB-DEX hydrogels possessed
higher water content than CB-DEX ones, facilitating the formation of larger ice
crystals during the lyophilization process.20,31 In contrast, the CB-DEX hydrogels with
lower water content tended to form smaller ice crystals, leading to smaller pore size
after lyophilization, which was consistent with the swelling capacity of the two
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zwitterionic hydrogels.
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Figure 6. The SEM images of the SB-DEX and CB-DEX hydrogels.
Zeta potential measurements
The surface charge of materials was reported to influence many properties,
including protein resistance and cell adhesion.32 Zwitterionic biomaterials are
neutrally charged as a whole, leading to better antifouling ability. The zeta potentials
of the hydrogels were measured to determine the surface charge of the ionic dextran
based hydrogels. As a control, the native dextran hydrogel was measured to be neutral
with a zeta potential of -0.03 mV. The SB-DEX and CB-DEX hydrogels had zeta
potentials of -0.58 mV and -0.53 mV, respectively, suggesting that the SB-DEX and
CB-DEX hydrogels are zwitterionic as expected.
ITC test
The ITC titration curves for dextran and SB-DEX were measured with a
fibrinogen solution titrant (Figure 7). It is observed that the generated heat during the
titration is different between dextran and SB-DEX solution at the same concentration.
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