Cationic hybrid hydrogels from amino-acid-based poly(ester amide): Fabrication, characterization, and biological properties

Article abstract of DOI:10.1002/adfm.201103147

A family of biodegradable, biocompatible, water soluble cationic polymer precursor, arginine-based unsaturated poly (ester amide) (Arg-UPEA), is reported. Its incorporation into conventional Pluronic diacrylate (Pluronic-DA) to form hybrid hydrogels for a

Full text of DOI:10.1002/adfm.201103147

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Cationic Hybrid Hydrogels from Amino-Acid-Based
Poly(ester amide): Fabrication, Characterization,
and Biological Properties
Jun Wu, Dequn Wu, Martha A. Mutschler, and Chih-Chang Chu*
systems, synthetic hydrogels have received
a lot of attention because they are more
reproducible, and can offer better control
over the structure, physical integrity and
mechanical properties.[1–6]
A family of biodegradable, biocompatible, water soluble cationic polymer
precursor, arginine-based unsaturated poly (ester amide) (Arg-UPEA), is
reported. Its incorporation into conventional Pluronic diacrylate (Pluronic-DA)
to form hybrid hydrogels for a significant improvement of the biological per-
formance of current synthetic hydrogels is shown. The gel fraction (Gf), equi-
librium swelling ratio (Qeq), compressive modulus, and interior morphology
of the hybrid hydrogels as well as their interactions with human fibroblasts
and bovine endothelial cells are fully investigated. It is found that the incor-
poration of Arg-UPEA into Pluronic-DA hydrogels significantly changes their
Qeq, mechanical strength, and interior morphology. The structure–property
relationship of the newly fabricated hybrid hydrogels is studied in terms
of the chemical structure of the Arg-UPEA precursor, i.e., the number of
methylene groups in the Arg-UPEA repeating unit. The results indicate that
increasing methylene groups in the Arg-UPEA repeating unit increases Qeq
and decreases the compressive modulus of hydrogels. When compared with
a pure Pluronic hydrogel, the cationic Arg-UPEAs/Pluronic hybrid hydrogels
greatly improve the attachment and proliferation of human fibroblasts on
hydrogel surfaces. A bovine aortic endothelial cells (BAEC) viability test in the
interior of the hydrogels shows that the positively charged hybrid hydrogels
can significantly improve the viability of the encapsulated endothelial cell
over a 2 week study period when compared with a pure Pluronic hydrogel.
For many pure (single component)
synthetic hydrogels, the lack of tunable
property/functionality and unsatisfied cel-
lular interaction greatly limits their bio-
medical applications. To solve these prob-
lems, hybrid hydrogels, which contain
two or more components (precursors),
have been developed. The introduction
of new functional precursors can help to
tune or bring new property/functionality,
such as charge property, hydrophobic/
hydrophilic property, pH and tempera-
ture responsive properties, and functional
groups.[7–19] For example, Chao et al. used
maleic chitosan to bring negative charge
property, carboxylic acid functional groups
and polysaccharide component into PEG
hydrogel.[16] Guo et al. and Pang et al.
incorporated hydrophobic phenylalanine
based poly (ester amide)s (Phe-PEA) into
PEG hydrogel to make the hydrogel more
hydrophobic and cell friendly.[17,18] Wu
et al. reported the integration of a cationic
hydrophilic precursor, 2-(acryloxy)ethyl]trimethylammonium
chloride precursor with a hydrophobic N-vinylcaprolactam pre-
cursor to achieve a hydrophilic-hydrophobic balanced hybrid
hydrogels for the delivery of ovalbumin.[19]
1. Introduction
In recent years, hydrogels have attracted many interests because
of their good biocompatibility, high water content, 3D micro-
porous structure, permeability for oxygen and nutrients, and
tissue-like elastic properties.[1–6] Among the reported hydrogel
In this study, we reported the development of a family of
cationic arginine based precursors (Arg-UPEA, Scheme 1) to
test the hypothesis that the introduction of cationic Arg-UPEA
into single-precursor-based hydrogels can improve their cellular
interactions because of the cationic property and biocompat-
ibility of Arg-UPEA. Pluronic hydrogel was selected as a model
synthetic hydrogel for testing our hypothesis because it has
been used in many reported studies.[20–24] Human fibroblast
and bovine endothelial cells were used to compare the cellular
response difference between the Pluronic hydrogel and Arg-
UPEA/Pluronic hybrid hydrogel in terms of cell attachment on
hydrogel surface and cell encapsulation inside the hydrogel.
Arg-UPEAs is a relatively new member of the amino acid
based poly (ester amide)s (AA-PEAs) family. Recently, AA-PEAs,
a newly developed biodegradable biomaterial family, have
shown very low cytotoxicity and muted inflammatory response,
Dr. J. Wu, Dr. D. Wu, Prof. C.-C. Chu
Department of Biomedical Engineering
Cornell University
Ithaca, NY, 14853-4401, USA
E-mail: cc62@cornell.edu
Dr. D. Wu, Prof. C.-C. Chu
Department of Fiber Science and Apparel Design
Cornell University
Ithaca, NY, 14853-4401, USA
Prof. M. A. Mutschler
Department of Plant Breeding
Cornell University
Ithaca, NY, 14853-4401, USA
DOI: 10.1002/adfm.201103147
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structures of these 4 monomers II were confirmed by FTIR
and 1HNMR, and the details were described and discussed
elsewhere.[38]
Four types of Arg-UPEA precursors differing in y number
were prepared according to the chemical scheme in Scheme 2.
The yield of the final product was high, i.e., >85%. The chemical
structure of Arg-UPEAs was confirmed by FTIR and H-NMR,
and their yields, water solubility, charge density, glass transition
temperature (Tg), molecular weight of repeating unit, molecular
weight are given in Table 1. The Arg-UPEAs were moisture sen-
sitive and should be stored in sealed bottles at 4 °C or lower
temperature in dark place.
For the solubility of Arg-UPEA in common organic solvents,
1.0 mg/mL was used as solubility criteria to judge whether a
polymer is soluble or insoluble at room temperature. Due to
their strong polar nature, Arg-UPEAs tended to dissolve in
polar solvents. All the synthesized Arg-UPEAs were soluble
in polar organic solvents such as DMSO, DMF and methanol,
but insoluble in non-polar or weak polar organic solvents such
as ethyl acetate, THF or chloroform. The effect of y material
parameters on Arg-UPEA water solubility (Table 1) revealed that
y had a major impact on the water solubility of Arg-UPEAs; and
an increase in the methylene chain length in the diols (y) part
reduced the water solubility significantly due to the increasing
Scheme 1. Chemical structure of unsaturated Arg-PEA (Arg-UPEA).
and support natural wound healing.[25–37] For this study, a type
of water soluble unsaturated AA-PEAs based on L-arginine (Arg)
(Arg-UPEA) have been prepared[38] to overcome the aqueous
solubility problem so that the hybrid hydrogels could be fabri-
cated in an aqueous medium which would be advantageous for
cell encapsulation or impregnation of biolog-
ically originated species such as proteins.
2. Results and Discussion
2.1. Preparation and Characterization
of Polymer Precursors
The Pluronic (F127), with an average mole-
cular weight (MW) of 12 600, was used
here to prepare one of the two precursors
for the photo-crosslinking. The synthesis of
F127-DA followed the previously reported
procedures.[23] The chemical structure of
F127-DA was confirmed by 1H-NMR and
Fourier transform infrared (FTIR) spectros-
copy. The final product was white powder
and can dissolve in distilled water to form a
clear and transparent solution at room tem-
perature or 4 °C. The final product should be
re-purified by dialysis before any biological
tests.
The unsaturated di-p-nitrophenyl esters
of dicarboxylic acids (NF) and tetra-p-
toluenesulfonic acid salts of bis (L-arginine)
alkylene diester monomers were synthesized
and characterized as previous reports.[28,30,38]
The only difference among the four types of
monomers II (Arg-2-S, Arg-3-S, Arg-4-S, and
Arg-6-S) prepared is the methylene chain
length (y) in the diol part between the two
adjacent ester groups: number of CH2 varies
from 2 (Arg-2-S) to 6 (Arg-6-S). The chemical Scheme 2. Synthesis of monomers and Arg-UPEA polymers.
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T a b le . 1 Chemical and physical properties of Arg-UPEAs.
of methylene groups in the y part of the Arg-UPEAs revealed
that an increase in y led to a lower Tg. The Tg decreased from
112 °C to 88 °C when the y value was increased from 2 to 6.
This reduction in Tg with increasing y (or x) was attributed to
the increasing chain flexibility and hence free volume.
2-UArg-2-S 2-UArg-3-S 2-UArg-4-S 2-UArg-6-S
Yield [%]
87
91
93
89
Charge Density [mol kg−1
]
2.50
798.9
2.46
812.9
2.42
826.9
2.34
855.0
Molecular Weight of
Repeating Unit [g mol−1
]
2.2. Fabrication and Characterization of Arg-UPEA/Pluronic-DA
Hybrid Hydrogels
Molecular Weight, Mn
12.93
14.01
14.56
16.14
15.71
17.49
14.82
16.33
[kg mol−1
Molecular Weight, Mw
[kg mol−1
]
In this study, Irgacure 2959 was used as the photo-initiator in
an aqueous system via the UV photomeans. The initiator was
previously reported to cause minimal toxicity (cell death) over a
broad range of mammalian cell types and species ranging from
human fetal osteoblasts to bovine chondrocytes.[39]
]
Polydispersity, Mw/Mn
1.08
112
1.11
103
1.11
94
1.10
88
Tg [°C]
Water Solubility [mg/mL]
20
2
30
2
10
1
2
0.5
All the fabricated Arg-UPEA/F127-DA hydrogels were trans-
parent after reaching their swelling equilibrium as shown in
Figure S1 (Supporting Information), which is an example of the
2-UArg-2-S/F127-DA hydrogel at the feed weight ratio 1/4. The
right image was a corresponding dried hydrogel. The swelling
behavior, compression modules and interior morphology of
these hybrid hydrogels were systematically examined as a func-
tion of the weight feed ratio and the y parameter in the Arg-
UPEA precursor. Table 2 summarizes the gel fraction (Gf),
compression modulus, equilibrium swelling ratio (Qeq) and ele-
mental analysis (N). The successful fabrication of Arg-UPEA/
F127-DA hybrid hydrogels was confirmed by the elemental
analysis. By measuring the nitrogen element percentage of
dried hydrogels, it was confirmed that most of the Arg-UPEA
was successfully crosslinked with F127-DA as F127-DA doesn’t
have any N element. The measured N contents in Table 2 were
slightly higher than the theoretical values (in the parentheses).
The gel fraction data (Gf) data showed that a pure F127-DA
hydrogel had a higher Gf value (more than 90%) than the Arg-
UPEA/F127-DA hybrid hydrogels (around 80%). The reason
for this Gf difference is that F127-DA has a higher activity than
Arg-UPEA. For the effects of y and feed ratio on Gf, the results
in Table 2 showed that y value has almost no effect on the Gf;
while the feed ratio of F127-DA to Arg-UPEA has some effect
on the Gf, and a higher feed ratio of F127-DA to Arg-UPEA
resulted in a higher Gf. The reason could be due to the weak
hydrophobicity. The Arg-UPEAs showed reduced water solu-
bility when compared to the reported saturated Arg-PEAs.[30,38]
The MW data in Table 1 indicated that all the Arg-UPEAs
had Mn between 12.5 kg mol−1 and 16.0 kg mol−1 with a narrow
polydispersity of 1.07–1.10. The y values did not have any sig-
nificant impact on MW and polydispersity of Arg-UPEAs. When
compared with saturated Arg-PEAs, the MW of the Arg-UPEAs
did not show any big difference and were in the same range.
For the thermal property of Arg-UPEAs, the melting point
(Tm) could not be detected, which was consistent with the
reported saturated Arg-PEAs.[38] The Tg’s of Arg-UPEAs (Table 1)
were in the range of 88–112 °C, which had the same range as the
Tg of phenylalanine-based unsaturated PEAs (Phe-UPEAs).[28]
Due to the presence of double bonds in the repeating unit of
the Arg-UPEA backbone, much higher Tg values were observed
when compared with the saturated Arg-PEAs.[38] For example,
2-UArg-2-S had a Tg around 112 °C, while 2-Arg-2-S had a Tg
52 °C, more than 100% increase in Tg by simply introducing
unsaturated C=C bond in the polymer backbone. Pang et al.
reported that the location of C=C bonds may also have a signifi-
cant effect on Tg.[32] For example, if the C=C bonds were located
in the side chain, the Tg value may decrease significantly.[32]
Similar magnitude of Tg change was also reported for the Phe-
based PEAs.[25,28] An examination of the effect of the number
T a b le 2 . Arg-UPEA/F127-DA hybrid hydrogels and their physicochemical properties.
a)
Sample
Weight Ratio
Gf
[%]
Qeq
Compressive Modulusa)
[KPa]
N
[%]
[%]
F127-DA
100:0
4:1
4:1
4:1
4:1
3:2
3:2
3:2
3:2
92
83
80
81
79
77
79
76
75
0.00 (0.00)
2.80 (2.61)
2.76 (2.54)
2.71 (2.55)
2.62 (2.50)
5.61 (5.40)
5.52 (5.44)
5.43 (5.27)
5.24 (5.10)
1144 17
1785 74
1843 77
1917 59
2158 98
2014 63
2191 94
2287 63
2407 117
12.27 0.34
4.85 0.41
4.43 0.23
3.77 0.24
2.58 0.25
1.97 0.21
1.91 0.11
1.63 0.19
0.77 0.17
F127-DA/2-UArg-2-S
F127-DA/2-UArg-3-S
F127-DA/2-UArg-4-S
F127-DA/2-UArg-6-S
F127-DA/2-UArg-2-S
F127-DA/2-UArg-3-S
F127-DA/2-U-Arg-4-S
F127-DA/2-UArg-6-S
a)
Q
eq
and compressive modulus were measured in DI water at room temperature.
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of 2014 63%, a 13% increase in Qeq. The reason could be due
to the relatively very high hydrophilicity of the Arg-UPEA pre-
cursor which could lead to a more loose 3D network structure
upon swelling, i.e., higher equilibrium swelling ratio with those
hybrid hydrogels having a higher percentage of the Arg-UPEA
components.
We further investigated the effect of molecular structure (y
value) of the Arg-UPEA on the swelling property of the hybrid
hydrogels at a constant weight feed ratio of Arg-UPEA to
F127-DA. As shown in Table 2, the swelling ratio increased as
the number of methylene groups in the repeating unit of Arg-
UPEA increased. For example, at the weight feed ratio of 1 to 4,
the equilibrated swelling ratio increased from 1785 74% from
2-UArg-2-S/F127-DA (1/4 w/w) to 2158 98% from 2-UArg-
6-S/F127-DA (2/3 w/w). Pang et al. also reported the similar
effect of the methylene chain length on hydrogel swelling in
their study of the Phe-based PEA and polyethylene glycol dia-
crylate hybrid hydrogels with.[17] They reported that a shorter
CH2 segment from C8 to C2 decreased the Qeq value slightly
from 860% to 750%.[17] They suggested that this relationship
between the shorter methylene chain length and lower hybrid
hydrogel equilibrium swelling could be due to the formation of
a tighter network structure from denser double bond contents
due to shorter methylene chain spacer between two adjacent
double bonds.[17]
Figure 1. Swelling kinetics of Arg-UPEA/Pluronic-DA hydrogels in distilled
water at room temperature.
crosslinking ability of Arg-UPEA precursor as it can’t form
hydrogels by itself. The upper limit of the weight feed ratio of
F127-DA to Arg-UPEA in the hybrid hydrogels for a successful
fabrication is 2:3 (60 wt% of Arg-UPEA) and the hydrogels
under this composition is very soft. Our unpublished data
showed the thermo-responsive property of the hybrid hydrogels
since Pluronic is thermoresposive (LCST behavior). This prop-
erty did affect the hydrogel properties, such as swelling ratios,
but did not affect the cellular interaction significantly. Since
it’s not the focus of this report, it will be discussed in another
report.
The equilibrium swelling ratio tests were performed in
deionized water (DI water) and the Qeq were also summarized
in Table 2. During the tests of equilibrium swelling ratio, the
swelling kinetics of hydrogels (how fast the hydrogel swells)
was also measured. Figure 1 showed the swelling kinetics for
a series of hybrid hydrogels as a function of the y parameter
of the Arg-UPEA precursor at room temperature in DI water.
Within first several hours, all the hydrogels showed very high
swelling rate, and these early stage swelling rates appear to be
independent of the composition of the hybrid hydrogels. After
that, the hydrogels’ swelling rates were much slower and will
eventually reach equilibrium state after around 20 h. These
swelling kinetics observed in the Arg-PEA/F127 hybrid hydro-
gels are consistent with other types of AA-PEA-based hybrid
hydrogel systems.[17,18] For example, both Pang et al. and Guo
et al. reported the swelling kinetics study of Phe-UPEA hybrid
hydrogels,[17,18] and within the first several hours, the Phe-
UPEA hybrid hydrogels showed very high swelling rate, and
then tapped off after 24 h.[17]
2.3. Mechanical Properties (Compressive Modulus)
of Arg-UPEA/F127-DA Hybrid Hydrogels
The mechanical property (compressive modulus) of the Arg-
UPEA/F127-DA hybrid hydrogels is summarized in Table 2.
The pure F127-DA has a compress modulus of 12.27 KPa and
the Arg-UPEA/F127-DA hybrid hydrogels showed much lower
compress moduli than a pure F127-DA hydrogel, in the range
between 0.77 KPa and 4.85 KPa. For the effect of molecular
structure (y value) of Arg-UPEA on the compressive modulus
of the hybrid hydrogels, it was found that the compressive
modulus decreased with an increase in y value (Table 2). For
example, at a fixed feed weight ratio of Arg-UPEA to F127-DA
of 1:4, an increase of y from 2 (2-UArg-2-S/F127-DA), 4
(2-UArg-4-S/F127-DA) to 6 (2-UArg-6-S/F127-DA) caused their
compressive moduli decreased from 4.85 0.41, 3.77 0.24
to 2.58 0.25 KPa, respectively. The compressive modulus of
Arg-UPEA/F127-DA hydrogel is much less than our previous
reported Phe-UPEA/PEG8000-DA system.[17,18] For example,
for the hybrid hydrogel system of Arg-UPEA/F127-DA with a
weight feed ratio of 1:4, its compressive moduli is in the range
of 2-5 KPa; while Phe-UPEA/PEG8000-DA at the same feed
ratio had the compressive moduli 560 KPa, about 100 times
of the Arg-UPEA/F127-DA system. Such a big difference is
mainly caused by the significant difference between Arg-UPEA
and Phe-UPEA precursors as the Arg-UPEA precursor is far
more hydrophilic and ionic than the hydrophobic and non-ionic
Phe-UPEA precursor used in the Guo et al. study.[28] Pang et al.
also discussed the compression modulus of some other types of
Phe-PEA hybrid hydrogels whose photo-reactive double bonds
were in the pendant group of the allyl glycine unit (AG)[17]
rather in the Phe-PEA backbone as in the Guo et al. study.[18]
The equilibrated swelling ratios of the hybrid hydrogels gen-
erally increase with an increase in either the weight feed ratio
of Arg-UPEA to F127-DA precursors or the y value in the Arg-
UPEA precursor. For example, 2-UArg-2-S/F127-DA (1/4 w/w)
had an equilibrated swelling ratio of 1785 74%, while 2-UArg-
2-S/F127-DA (2/3 w/w) had a higher equilibrated swelling ratio
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these Arg-UPEA/F127-DA hybrid hydrogels
(Figure 2B–D) have larger average pore size
and thinner cell wall. For example, a pure
F127-DA hydrogel had an average pore size
8.0-10.0 μm, while all Arg-UPEA/F127-DA
hybrid hydrogels had the average pore size
10.0-15.0 μm. One of the distinctive mor-
phology of these Arg-UPEA/F127-DA hybrid
hydrogels from a pure F127-DA hydrogel
was that the Arg-UPEA/F127-DA hybrid
hydrogels showed some nano-size fiber webs
entangled with the cells. At a fixed feed ratio
of the 2 precursors, no significant difference
of the pore size was observed among the Arg-
UPEA/F127-DA hybrid hydrogels regardless
of the difference of y material parameter in
the Arg-UPEA precursors. Guo et al. and
Pang et al. also reported the SEM images of
the interior morphology of other AA-PEA
hybrid hydrogels.[17,18] Compared with Guo
and Pang’s AA-PEA hybrid hydrogel systems,
the Arg-UPEA/F127-DA hybrid hydrogels
studied here showed some unique nano-size
fiber webs entangled with the cells, while
Guo and Pang’s did not. All the prepared Arg-
UPEA/F127-DA hybrid hydrogels are biode-
gradable, Figure S2 (Supporting Information)
Figure 2. SEM images of Arg-UPEA/F127-DA hybrid hydrogels at weight feed ratio of Arg-
UPEA/F127-DA of 1/4. A) Pure F127-DA hydrogel, B) 2-UArg-2-S/F127-DA hybrid hydrogel,
C) 2-UArg-4-S/F127-DA hybrid hydrogel, and D) 2-UArg-6-S/F127-DA hybrid hydrogel. The
scale bar is 5 μm in (A–D).
Pang et al. reported that the compressive modulus of their Phe-
PEA hybrid hydrogels increased with the increasing of the AG
(allyl glycine) contents in the Phe-PEA-AG precursor.[17] As we
know, the mechanical property of hydrogels was very important
for their interaction with cells.[2] This Arg-UPEA/Pluronic-DA
hybrid hydrogel system offered a variety of mechanical prop-
erty choices for tissue engineering applications by adjusting the
material parameter of the precursor as well as its feed ratio to
the co-precursors.
showed an example of the interior morphology change of Arg-
UPEA/F127-DA hybrid hydrogel after 60 days of degradation.
2.5. Human Fibroblast Cell Attachment
and Proliferation on Hydrogel Surfaces
To study the cellular interaction with Arg-UPEA/F127-DA
hybrid hydrogels, the Detroit 539 human fibroblast cells were
cultured on the surface of Arg-UPEA/F127-DA hybrid hydro-
gels to investigate the cell attachment and proliferation per-
formance. Detroit 539 human fibroblast cells cultured onto
the 24 well cell culture plate without any other treatment were
used as the blank control, and fibroblast cells cultured on
the pure F127-DA hydrogel was used as a hydrogel control.
Figure 3 showed a representative example of the Detroit 539
human fibroblast cells cultured on the surface of 2-UArg-2-S/
F127-DA (1/4 w/w) hybrid hydrogel. When compared with the
2.4. Interior Morphology (SEM) of Arg-UPEA/F127-DA
Hydrogels
To further understand the 3D structure of Arg-UPEA/F127-DA
hybrid hydrogels, the cross-sectional interior morphology of
the hybrid hydrogels was examined and shown in Figure 2.
When compared with a pure F127-DA hydrogel (Figure 2A),
Figure 3. Representative microscopy images of Detroit 539 human fibroblast cells after 48 h of culture, 10×: A) cells cultured in 24 well cell culture
plate without any treatment (control), B) cells cultured on the surface of pure F127-DA hydrogel, and C) cells cultured on the surface of 2-UArg-2-S/
F127-DA(1/4, w/w) hydrogel.
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pure F127-DA hydrogel (Figure 3B) control, the hybrid hydrogel
(Figure 3C) had much higher amounts of the attached/prolifer-
ated fibroblast cells, and the amounts of the attached/prolifer-
ated fibroblast cells were slightly better than the blank control
(Figure 3A). From the aspect of the fibroblast cell morphology,
no significant morphology change was observed between the
hybrid hydrogel surface (Figure 3C) and the blank control
(Figure 3A) after 48 h of culture. There were no visible signs
of cell dying or unhealthy cells. The Detroit 539 human fibrob-
last cells attached onto the pure F127-DA hydrogel surface,
however, did show some morphology change (Figure 3B). The
data in Figure 3B show that the Detroit 539 human fibroblast
cells did not completely attach and spread on the F127-DA
hydrogel surface. Therefore, the introduction of Arg-UPEA to
Pluronic-DA hydrogel can significantly enhance the hydrogel’s
cell attachment and proliferation.
These qualitative cell morphological data were also confirmed
by the quantitative MTT assay for the attached/proliferated fibrob-
last cells (Figure S3, Supporting Information). Both Figure 3 and
Figure S3 (Supporting Information) showed that the blank and
the hybrid hydrogel have similar amounts of the attached Detroit
539 human fibroblast cells and both had significantly higher
amounts of attached cells than the pure F127-DA hydrogel.
The possible reasons for the observed encouraging Detroit
539 human fibroblast cell attachment and proliferation data
onto the 2-UArg-2-S/F127-DA hybrid hydrogel system can be
attributed to the excellent biocompatibility and cationic nature of
the Arg-UPEA component rather than the F127. Many reported
studies have used F127 or F127 derivatives to fabricate hydrogels
for tissue engineering applications.[7–15] However, those reported
studies indicated that F127 hydrogel itself can not support the cell
attachment/proliferation well, and many chemical modification
methods have been applied to F127 hydrogel
or PEG hydrogel system[40] showed the highest support of 3T3
fibroblast attachment and spreading than either RGD modified,
anionic, neutral or unmodified HEMA and PEG hydrogels in the
presence or absence of serum, Unfortunately, no 3T3 fibroblast
attachment and proliferation data on tissue culture plates (TCP)
were given in the Schneider et al. study to draw a conclusion
whether their cationic HEMA or PEG hydrogel systems would
exhibit the similar fibroblasts attachment and proliferation as the
TCP control as we demonstrated in our Arg-UPEA/Pluronic-DA
hydrogel system in Figure 3. Hynes et al. discussed how to uti-
lize the PEG/polylysine hydrogel scaffold to improve the survival
and promote the differentiation of neural stem cells.[41,42] These
excellent cell attachment and proliferation performances of Arg-
UPEA/F127-DA hybrid hydrogels suggest they may have a great
potential as a new type of scaffolds for various biomedical appli-
cations. Of course, these cationic Arg-UPEA/Pluronic-DA hybrid
hydrogel systems can be further improved with cell-adhesive lig-
ands for additional enhanced cell adhesion.
2.6. BAEC Cell Viability Inside Arg-UPEA/F127-DA Hybrid
Hydrogels
In order to understand the effects of Arg-UPEA on the long-
term encapsulated cell behavior, we tested the long term BAEC
cell viability encapsulated within the Arg-UPEA/F127-DA
hybrid hydrogel for 2 weeks at 37.0 °C and 5.0% CO2. The live-
dead assay was used to evaluate the BAEC cell viability and
was performed according to the manufacturer protocol (LIVE/
DEAD Cell Viability Assay Kit from Invitrogen). As shown in
Figure 4, after 2 weeks almost all of the remaining encapsu-
lated BAEC cells within 2-UArg-2-S/F127-DA hybrid hydrogel
system to improve the cellular interaction of
F127 hydrogels.[7–15] For example, Park et al.
reported using chitosan-F127 hydrogels for
cartilage regeneration.[7] Jung et al. reported
using TGF-β1-conjugated biodegradable
Pluronic F127 hydrogel for adipose-derived
stem cells culture.[13] Lippens et al. reported
using alanine modified F127 hydrogel for
mesenchymal stem cells culture.[15] Lin et al.
reported using poly(dimethyl siloxane-ure-
thane)/Pluronic F127 for L929 fibroblast cell
culture,[10] and their cell morphology data
indicated that the attachment performance
of L929 fibroblast on F127/poly(dimethyl
siloxane-urethane) hybrid hydrogels was con-
sidered non-cytotoxic, but still exhibited lesser
L929 fibroblast confluence from the cell cul-
ture plate negative control.
The significantly better fibroblast attach-
ment and proliferation on a cationic charged
hydrogel system as observed in the Arg-UPEA/
F127 hydrogel system is consistent with other
reported positively charged hydrogel sys-
tems, such as chemically modified PEGDA
Figure 4. Live-dead assay for BAEC cells encapsulated in the pure F127-DA hydrogel (A,B) and
2-UArg-2-S/F127-DA hybrid hydrogel (C,D). Green dots in the (A,C) are for the living cells and
red dots in (B,D) are for the dead cells
and HEMA hydrogels.[40–42]Schneider et al.
reported that the positively charged HEMA
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Unsaturated di-p-nitrophenyl ester of dicarboxylic acid (monomer
I, di-p-nitrophenyl fumarate (NF)) was prepared by reacting fumaryl
chloride with p-nitrophenol as previously reported.[28] Four types of
p-toluenesulfonic acid salt of L-arginine diesters (monomer II) were
prepared in this study: tetra-p-toluenesulfonic acid salt of bis(L-arginine)
ethane diesters Arg-2-S (y = 2); tetra-p-toluenesulfonic acid salt of bis-
(L-arginine) propane diesters, Arg-3-S (y = 3); tetra-p-toluenesulfonic
acid salt of bis(L-arginine) butane diesters, Arg-4-S (y = 4); tetra-p-
toluenesulfonic acid salt of bis(L-arginine) hexane diesters, Arg-6-S (y = 6).
y indicates the number of methylene group in the diol segment of the
Arg-UPEA. Arg-UPEAs were subsequently prepared by the solution
polycondensation of (I) and (II) monomers (NF and Arg-2-S, Arg-3-S,
Arg-4-S, or Arg-6-S) at different combinations (Scheme 2 and Table
S1, Supporting Information). The resulting Arg-UPEAs are labeled as
x-UArg-y-S, where x and y are the number of CH2 groups in diacid and
diol segments, respectively, and U means the Arg-PEA is unsaturated.
x was fixed at 2 because only one type of unsaturated monomer I (NF)
was used. The Arg-UPEAs synthesized with different combinations of
diacids and diols building blocks are: 2-UArg-2-S, 2-UArg-3-S, 2-UArg-4-S
and 2-UArg-6-S (Table S1, Supporting Information).
Fabrication of Arg-UPEA/Pluronic-DA Hybrid Hydrogels: Arg-UPEA/
Pluronic-DA hybrid hydrogels were prepared by the photo-polymerization
of two precursors (Arg-UPEA and Pluronic-DA) at different weight feed
ratios in an aqueous solution with a water soluble photo-initiator. The
Arg-UPEA and Pluronic-DA precursors were purified first by dissolving
the precursors in distilled water and were dialyzed against deionized
water (molecular weight cut off 4000) for 2 days. After that, the solutions
were lyophilized for 3 days using a Virtis Freeze Drier (Gardiner, NY)
under vacuum at −48 °C. An example of the fabrication of a hybrid
hydrogel was given below: 2-UArg-2-S (80.0 mg) and F127-DA (320.0 mg)
(1/4 weight ratio of 2-UArg-2-S/F127-DA) were added into a glass vial
and dissolved in deionized water (2.0 mL) to form a clear homogeneous
solution with light yellow color. Photoinitiator Irgacure 2959 (4.0 mg
which was 1.0 wt% of the total amounts of precursor) was added
into the precursors’ solution and dissolved completely. The precursor
(1:4 weight feed ratio) and the pure F127-DA hydrogel were
viable and healthy (stained in green, Figure 4A,C), but the pure
F127-DA hydrogel shows a lot of less green dot density but a lot
of unhealthy or dead cells (stained in red, Figure 4B), while the
2-UArg-2-S/F127-DA hybrid hydrogel shows far higher green
dot density and hardly any red dots (Figure 4C,D), The green
dots of different brightness and sizes mean that the living
BAEC cells were at the different depth of the 3D hydrogel. This
finding indicates that the incorporation of the cationic Arg-
UPEA moiety into F127 system could significantly facilitate the
proliferation and long-term viability of the encapsulated cells
within the 3D hydrogel interior.
3. Conclusions
A new family of cationic charged biocompatible Arg-UPEA/
Pluronic-DA hybrid hydrogel was successfully fabricated in
an aqueous medium via UV photocrosslinking with a photo-
initiator. The physicochemical, swelling, mechanical, and
morphological properties of the resulting hybrid hydrogels
were investigated. The newly designed and fabricated Arg-
UPEA/Pluronic-DA hybrid hydrogel system can offer many
advantages in terms of water soluble precursors for easier
pre-loading of cells or therapeutic proteins, cationic charge
for better cell attachment and proliferation, mechanical prop-
erty, a range of hydrophobicity vs. hydrophilicity balance, and
pore size. We demonstrated that by varying the feed ratio of
Arg-UPEA to Pluronic-DA and the type of Arg-UPEA, we
can finely tune the swelling, mechanical, and morphological
properties of such a cationic hybrid hydrogel system. These
results contribute to the understanding of the structure–prop-
erty relationship of Arg-UPEA. Cell culture studies indicated
that the incorporation of Arg-UPEA to Pluronic-DA hydrogel
significantly increase the cell attachment, proliferation, and
viability of both Detroit 539 human fibroblasts and bovine
aortic endothelial cells.
solution was then transferred to
a custom-made 20 well Teflon
mold (diameter 12.0 mm and depth ≈ 4.0 mm of each well) using
a micropipette. The precursor solution in the molds was irradiated
by a long-wavelength UV lamp (365 nm, 100 W) for specified time
(5.0 min) at room temperature. The irradiation distance was 5.0–10.0 cm.
The resultant hydrogels were moved from the mold and immersed in
distilled water at room temperature for 48 h to remove any residual
chemicals. The distilled water was replaced periodically. After this
purification process, the hydrogel was soaked in distilled water to reach
swelling equilibrium, and dried in vacuum at room temperature for 48 h
before further characterization and application. The gel fraction (Gf)
of the resulting hydrogels was calculated by the following equation:
Gf = (Wd/Wp) ×100%, where Wd is the weight of dry hydrogel and Wp is
the total weight of the two precursors and the photo-initiator.
4. Experimental Section
Materials: Pluronic (F127, MW 12 600 and 70% PEG content), acryloyl
chloride, triethylamine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) were purchased from Sigma-Aldrich
(St. Louis, MO) and used without further purification. 2-Hydroxy-l-
[4-(hydroxyethoxy)phenyl]-2-methyl-l-propanone (Irgacure 2959) was
donated by Ciba Specialty Chemicals Corporation. L-Arginine (L-Arg),
p-toluenesulfonic acid monohydrate, fumaryl chloride, ethylene glycol,
1,3-propanediol, 1, 4-butanediol, 1,6-hexaniol and p-nitrophenol were
all purchased from Alfa Aesar (Ward Hill, MA) and used without further
purification. Other chemicals and reagents if not otherwise specified
were purchased from Sigma-Aldrich (St. Louis, MO).
Synthesis of Hydrogel Precursors: Pluronic-DA (F127-DA) was
synthesized according to a previously reported method[23] (Scheme S1,
Supporting Information). The Arg-UPEAs were synthesized by the same
procedures reported before,[25,28,30] except the unsaturated diacids were
used to provide unsaturated moiety in the AA-PEA backbone. Briefly, the
synthesis of Arg-UPEAs can be divided into the following three steps:
the preparation of unsaturated di-p-nitrophenyl ester of dicarboxylic
acid (I); the preparation of tetra-p-toluenesulfonic acid salts of bis(L-
arginine), α,ω-alkylene diesters (II); and the synthesis of Arg-UPEAs (III)
via solution polycondensation of (I) and (II).
Hydrogel Equilibrium Swelling Ratio: The equilibrium swelling ratio
(Qeq) of hydrogels was calculated by the following equation: Qeq
=
[(We – Wd)/Wd]×100%, where Ws is the weight of a swollen hydrogel at
equilibrium and Wd is the weight of the corresponding dry hydrogel at
t = 0. All swelling ratio results were obtained from triplicate samples and
data were expressed as the means standard deviation.
Compressive Modulus Measurement by Dynamic Mechanical Analyzer
(DMA): The mechanical property of the Arg-UPEA/F127-DA hydrogels
was measured by a DMA 2980 Dynamic Mechanical Analyzer (TA
Instruments Inc., New Castle, DE) in a controlled force mode (CF-mode).
The swollen hydrogel samples in circular disc shape were submerged in
distilled water and mounted between the movable compression clamp
(diameter 30.0 mm) and the fluid cup with a 0.1 N preloading force. A
force ramp from 0.1 N at a rate of 0.3 or 0.5 N.min−1 was applied. All
measurements were carried out at room temperature. The compression
elastic modulus (E) of the swollen hydrogel was extracted by plotting
the compressive stress versus strain. All compression elastic modulus
data in this study were obtained from triplicate samples and data were
expressed as the mean standard deviation.
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DOI: 10.1002/adfm.201103147
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Cell Attachment and Proliferation on Arg-UPEA/F127-DA Hybrid
Hydrogels Surface: The cell attachment and proliferation on the Arg-UPEA/
F127-DA hybrid hydrogel surfaces was evaluated by cell morphology.
Pure F127-DA hydrogel was chosen as the hydrogel control, and the cell
culture plate without any treatment was used as the blank control. The
cells used for this study were Detroit 539 human fibroblasts. The purified
hydrogels were cut into round shape with the diameter that just filled the
well of 24-well cell culture plates. The hydrogels were sterilized under UV
light (from the cell culture hood) for 1.0 h. After that, the hydrogels were
washed twice by PBS buffer and cell culture media. Then, the hydrogels
were placed into the wells of cell culture plate and fixed by sterilized
rubber ring which has the same diameter as the well of cell culture plate.
Detroit 539 human fibroblasts were seeded at an appropriate cell density
(10,000 cells/well) and incubated overnight. After incubation (48 h), the
cell attachment and proliferation on the hydrogel surface was record
by an optical microscope. For MTT assay, the attached cells were first
detached from the hydrogel surface or cell culture plate surface by trypsin
(0.1 mg mL−1 ) treatment, then the detached cells were transferred into
a new 96 cell culture place, after incubation (12 h), the MTT assay were
processed.
cut and fixed on aluminum stubs and then coated with gold for 30 s for
interior morphology observation with a scanning electron microscope
(Leica S440, Germany).
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
This study was supported in part by a seed grant from Cornell University
Ithaca - Weill Intercampus Program. The authors also thank Prof.
C. A. Reinhart-King at Cornell University for using her lab for some cell
culture work. The molecular weights of Arg-UPEAs were obtained with
the help of MediVas, LLC.
Cell Viability inside Arg-UPEA/F127-DA Hybrid Hydrogels: In order to
test the cell viability inside the Arg-UPEA/F127-DA hybrid hydrogels, the
BAECs were encapsulated into hydrogels by the following steps: Purified
hydrogel precursors and initiators were dissolved in a PBS buffer, and
then the cells (107 mL−1 in media), FBS, antibiotics and other nutrients
of complete cell culture media were added. The final mixture solution
has 20.0 wt% precursors, 60 000 cells mL−1 , and 10.0 wt% FBS inside.
All other components of complete cell culture media in the mixture
have the exactly same concentration as the normal BAEC cell culture
media recommended by ATCC. The mixture were injected into 24-well
cell culture plate (0.5 mL per well) by a pipette and crosslinked under
irradiation (100 W UV) for 5 min and the irradiation distance is 5 cm.
After crosslinking, complete cell culture media (0.5 mL) was added into
each well. The BAEC-loaded hydrogels were incubated for 2 weeks at
37 °C, 5% CO2. Cell culture media was changed every other day. The live-
dead assay was then performed according to the manufacturer protocol
(LIVE/DEAD Cell Viability Assay Kit from Invitrogen).
Received: December 27, 2011
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Adv. Funct. Mater. 2012,
DOI: 10.1002/adfm.201103147
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