High viability of cells encapsulated in degradable poly(carboxybetaine) hydrogels

Article abstract of DOI:10.1021/la303390j

In this study, we report a degradable poly(carboxybetaine) (pCB) hydrogel, produced via a thiol-disulfide exchange reaction for cell encapsulation. A pCB dithiol was synthesized as a cross-linker and reacted with a pyridyl dithiol-containing CB copolymer to form a hydrogel. We evaluated the biocompatibility of the pCB-based hydrogel via encapsulation of three cell types, including NIH3T3 fibroblasts, MG63 osteoblast-like cells, and HepG2 hepatocarcinoma cells. Up to 90% of cells retained their viability in the pCB hydrogel even at low cell-seeding densities under serum-free conditions after a 9-day culture. Results are compared with a degradable poly(ethylene glycol) methacrylate (PEGMA) hydrogel, which showed very low cell viability under serum-free condition after a 3-day culture. We incorporated an RGD peptide into the CB hydrogel using a cysteine-terminated cross-linker, which was shown to promote cell proliferation.

Full text of DOI:10.1021/la303390j

Article
pubs.acs.org/Langmuir
High Viability of Cells Encapsulated in Degradable
Poly(carboxybetaine) Hydrogels
Hsiu-Wen Chien,†,‡,§ Xuewei Xu,†,§ Jean-Rene Ella-Menye,† Wei-Bor Tsai,*,‡ and Shaoyi Jiang*,†
†Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States
‡Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan
S
* Supporting Information
ABSTRACT: In this study, we report a degradable poly(carboxybetaine)
(pCB) hydrogel, produced via a thiol−disulfide exchange reaction for cell
encapsulation. A pCB dithiol was synthesized as a cross-linker and reacted
with a pyridyl dithiol-containing CB copolymer to form a hydrogel. We
evaluated the biocompatibility of the pCB-based hydrogel via encapsula-
tion of three cell types, including NIH3T3 fibroblasts, MG63 osteoblast-
like cells, and HepG2 hepatocarcinoma cells. Up to 90% of cells retained
their viability in the pCB hydrogel even at low cell-seeding densities under
serum-free conditions after a 9-day culture. Results are compared with a
degradable poly(ethylene glycol) methacrylate (PEGMA) hydrogel, which
showed very low cell viability under serum-free condition after a 3-day
culture. We incorporated an RGD peptide into the CB hydrogel using a
cysteine-terminated cross-linker, which was shown to promote cell
proliferation.
growth.10,11 Hydrogels have been attractive candidates for
tissue-engineering scaffolds due to their tissue-like elasticity and
1. INTRODUCTION
Zwitterionic materials have received increasing attention due to
high water content, mimicking the interstitial tissue environ-
their importance in recent years. Recently, a series of water-
ment. Poly(ethylene glycol) (PEG)-based hydrogels have been
soluble zwitterionic compounds, such as carboxybetaine (CB)
the most widely used synthetic materials for cell encapsula-
monomers and cross-linkers, has been developed.1 Surfaces
tion10−13 because they provide a highly swollen environment
grafted with CB-based polymers have been demonstrated to be
similar to native tissues and possess a low level of nonspecific
ultralow fouling, which is defined as less than 5 ng/cm2
protein adsorption.13 Direct cell encapsulation during hydrogel
adsorbed proteins.2 These surfaces are highly resistant to
formation in situ is a popular strategy, but a minimally invasive
nonspecific protein adsorption, even from undiluted blood
plasma and serum, and prohibit long-term bacterial coloniza-
operation is required.11 The requirements for such a strategy
tion for up to 10 days.1 The ultralow-fouling properties of
include the nontoxicity of hydrogel materials, including their
precursors and final products, and an innocuous process of cell
zwitterionic materials result from high hydration around their
opposing charges via electrostatically induced hydration and the
encapsulation.10 One popular method is using diacrylate- or
high energy barrier required to remove hydration layers.3 Due
dimethacrylate-containing molecules that are cross-linked via a
free radical polymerization to make hydrogels.12 The toxic
to the high hydration and ultralow-fouling properties of
photoinitiator or radiation treatment can cause high cellular
zwitterionic materials, they have found a variety of biomedical
damage during in situ cell encapsulation. In order to reduce
applications. For example, proteins conjugated to a CB polymer
toxicity and free radical damage, macromolecules with various
are able to maintain their stability without sacrificing binding
affinity,4 and CB polymer-based drug delivery carriers with
end group functionalities, such as thiols, as used in this work,
are versatile for hydrogel formation.13,14
encapsulated drugs are very stable.5 CB-based hydrogels
prepared from a CB monomer and a CB cross-linker have
several advantages:6,7 very low protein adsorption and cell
adhesion, functionalizability, and excellent mechanical proper-
ties. Hydrogels with chemical and mechanical gradients have
also been developed.8 Recently, we demonstrated that cells that
were directly encapsulated in biodegradable and RGD-
functionalized CB hydrogels displayed a tissue-like morphology
after 4-week culture.9
In this study, we prepared a degradable pCB hydrogel as a
cell encapsulation material via a thiol−disulfide exchange
reaction (Figure 1). The disulfide-cross-linked hydrogel,
which is cleavable chemically by biological reductants such as
glutathione (GSH) or cysteine, is of particular interest as an
implantable scaffold due to its potential degradability. We
Received: August 22, 2012
Revised: November 9, 2012
Published: November 19, 2012
Three-dimensional (3D) scaffolds in tissue engineering serve
as a temporary support for cell accommodation and
© 2012 American Chemical Society
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Figure 1. Illustration of the formation of pCB (a) and RGD-pCB (b) hydrogels by the thiol−disulfide exchange reaction. (c) pCB hydrogel was
made from 7% (w/v) of pCB-PDP and 2% (w/v) of pCB-DT in PBS.
consisted of DMEM, 1% nonessential amino acid, 1 mM sodium
pyruvate, and 1% PS. All water used had been purified to 18.2 mΩ on
a Millipore Simplicity water purification system.
synthesized a pCB dithiol (pCB-DT) as a cross-linker; this
reacts with a pyridyl dithiol-containing CB copolymer (pCB-
PDP) to form a hydrogel. We evaluated the biocompatibility of
the pCB-based hydrogel via several types of cell encapsulation.
Finally, we incorporated a RGD peptide into the pCB hydrogel
via a cysteine-terminated cross-linker and evaluated cell
proliferation within the pCB hydrogel and RGD-pCB hydrogel.
2.2. Synthesis of Poly(CB-co-PDPMA) (pCB-PDP). N-(3-(2-
(Pyridyldithio)propanamido)ethyl methacrylate (PDPMA) was syn-
thesized as described in Scheme 1. 2-Aminoethyl methacrylate
hydrochloride (AEMA) (26.5 mg, 0.160 mmol) was dissolved in 5
mL of dichloromethane. N-Succinimidyl 3-(2-pyridyldithio) propio-
nate (SPDP) (50 mg, 0.16 mmol) and triethylamine (0.022 mL, 0.160
mmol) were added to the solution, which was stirred at room
temperature for 60 min. The reaction was diluted with dichloro-
methane, silica gel (5 g) was added to the solution, and the solvent was
removed under vacuum on the rotary evaporator. Solid sample was
purified via silica gel automatic column chromatography using a
gradient of dichloromethane:methanol (20:1 to 1:1). The pure
product was obtained as a thick colorless oil (50.0 mg, 0.153
2. EXPERIMENTAL SECTION
2.1. Chemicals. 2-Carboxy-N,N,-dimethyl-N-(2′-
(methacryloyloxy)ethyl) ethanaminium inner salt (carboxybetaine
methacrylate, CBMA) and (3-acryloylamino-propyl)-(2-carboxy-
ethyl)-dimethylammonium(carboxybetaine acrylamide, CBAA) were
synthesized using a previously published procedure.15,16 4-Cyano-4-
(phenylcarbonothioylthio)pentanoic acid (CTP), diethylene glycol
(DEG), N,N′-dicyclohexylcarbodiimide (DCC, 99%), 4-
(dimethylamino)pyridine (DMAP), fluorescin diacetate (FDA), poly-
(ethylene glycol) methacrylate (PEGMA, Mn = 360), poly(ethylene
glycol) dithiol (PEG-DT, Mn = 3400), propidium iodide (PI),
phosphate-buffered saline (PBS), triethylamine (TEA), dichloride
methane (DCM), methanol, chloroform, ethyl ether, and dioxane were
all purchased from Sigma-Aldrich. 2-Aminoethyl methacrylate hydro-
chloride (AEMA) and 4,4′-azobis(4-cyanovaleric acid) (V-501) were
purchased from Acros. 2,2′-Azobis(4-methoxy-2.4-dimethyl valeroni-
trile) (V-70) was purchased from Wako Chemicals. N -Succinimidyl 3-
(2-pyridyldithio)propionate (SPDP) was from ProteoChem. Cys-Gly-
Arg-Asp-Ser-Gly-Cys (CGRGDSGC) peptide was purchased from
Synthetic Biomolecules. The culture medium for L929 cells and MG63
cells contained Dulbecco’s Modified Eagle Medium (DMEM) with 1%
penicillin-streptomycin (PS). The culture medium for HepG2 cells
1
mmol). Yield: 96%. H NMR (CDCl3, 300 MHz); δ 8.47 (d, 1H, J =
5.1 Hz), 7.64 (m, 2H), 7.13 (m, 1H), 6.89 (bs, 1H), 6.10 (s, 1H), 5.58
(s, 1H), 4.29 (t, 2H, J = 5.5 Hz), 3.63 (m, 2H), 3.09 (t, 2H, J = 6.7
Hz), 2.64 (t, 2H, J = 6.7 Hz), 1.93 (s, 3H).
pCB-PDP was synthesized by a reversible addition−fragmentation
chain transfer (RAFT) polymerization according to the process briefly
described as follows (Scheme 1). In a 25 mL ampule, CBMA
monomer (1011 mg, 4.417 mmol), PDPMA monomer (60 mg, 0.184
mmol), CTP (5.14 mg, 0.0184 mmol), and V-501 (1.03 mg, 0.004
mmol) were dissolved in methanol (5.0 mL). The ampule contents
were purged with nitrogen for 1 h, and then the ampule was placed in
a preheated oil bath at 70 °C. The reaction was terminated after 24 h
by cooling the reaction tube in an ice bath followed by exposure to air.
Product was purified by dialysis against water and collected by
lyophilization, and the product yield was 56%. pPEGMA-PDP was
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equipped with a magnetic stirring bar, CTP (2.02 g, 7.2 mmol), DEG
(0.32 g, 3.0 mmol), and DMAP (0.73 g, 6.0 mmol) were dissolved in
10 mL of anhydrous chloroform. After the solution was homogenized
by stirring, the flask was placed in an ice bath. DCC (3.09 g, 15.0
mmol) was then added in portions. After 4 h of stirring at 0 °C, the
reaction mixture was allowed to warm to room temperature and stirred
overnight. Precipitated dicyclohexylurea was removed by filtration to
yield a clear red solution. After removal of the volatiles in vacuo, the
1.05 g of crude product was purified by column chromatography on
silica gel with a mobile phase of ethyl acetate/hexane (1/3, v/v). Yield:
Scheme 1. Synthetic Route of pCB-PDP
1
55.4%. H NMR (CDCl3): δ 1.94 (s, −S−C(−CN, −CH2−)−CH3);
2.41−2.78 (m, 8H, C−CH2−CH2−(CO)−); 3.75 (t, 4H, −(C
O)−O−CH2−CH2−O−); 4.28 (t, 4H, −(CO)−O−CH2−CH2−
O−); 7.42, 7.58, 7.94 (m, 10H, aromatic ring).
In a 25 mL ampule, CTP-DEG-CTP (0.189 g, 0.3 mmol) and
CBAA monomer (1.93 g, 9.0 mmol) were dissolved in methanol (5.0
mL) and the solution pH was adjusted to 4.5. V-70 (42.0 mg, 0.15
mmol) dissolved in methanol (1.0 mL) was then added. The solution
was stirred until all of the CTP-DEG-CTP was dissolved. The ampule
contents were purged with nitrogen for ∼1 h, and then the ampule was
placed in a preheated oil bath at 70 °C. The reaction was terminated
after 8 h by cooling the reaction tube in an ice bath followed by
exposure to air. The product (CTP-pCB-CTP) was purified by dialysis
against water (pH 4−5) and isolated by lyophilization. The calculated
average molecular weight (Mn) is 4500. Yield: 1.20 g, 56.6%. 1H NMR
(CD3OD): δ 1.92 (s, −S−C(−CN, −CH2−)−CH3); 3.76 (t, −(C
O)−O−CH2−CH2−O−); 4.30 (t, −(CO)−O−CH2−CH2−O−);
7.46−8.02 (m, aromatic ring); 1.36−1.76 (b, −(CH−CH2)n−); 1.88−
2.08 (b, −(CH−CH2)n− and −CH2−CH2−CH2−); 2.56−2.66 (t,
−CH2−COO−); 3.02−3.12 (b, CH3−N+−CH3); 3.14−3.34 (b,
−CH2−CH2−CH2−); 3.50−3.59 (t, N+−CH2−CH2−COO−).
synthesized following the same procedure as illustrated in Scheme S1,
Supporting Information.
2.3. Synthesis of Thiol-Functionalized pCB (pCB-DT). Difunc-
tional chain transfer agent (CTP-DEG-CTP) was synthesized as
described in Scheme 2. In a 50 mL one-neck round-bottom flask
Scheme 2. Synthetic Route of pCB-DT
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Thiol-functionalized polymer (pCB-DT) was harvested by cleaving
dithioester end groups in CTP-pCB-CTP. CTP-pCB-CTP (0.45 g, 0.1
mmol) and methanol (10 mL) were added to a 25 mL round-
bottomed flask equipped with a magnetic stir bar. The mixture was
stirred until a homogeneous solution was obtained. To this solution
was added propylamine as a solution in methanol (2.0 M) (1.00 mL,
2.0 mmol). The resulting solution was stirred at room temperature for
5 h. Subsequently, the solution was loaded to a dialysis bag and
dialyzed against water (pH 4−5) for 2 days. Crude product was
isolated by lyophilization. Polymer was then dissolved in methanol (10
mL), and then DTT (0.154 g, 1.0 mmol) was added to the solution
and allowed to stir at room temperature under N2 atmosphere for 6 h.
The resulting thiol-functionalized polymer (pCB-DT) was obtained as
a white powder by precipitation into cooled ether, followed by drying
molecular weights of the copolymers (pCB-PDP) are
summarized in Table 1. The ratio of PDPMA to copolymer
Table 1. Synthesis of pCB-PDP
in feed (mole
fraction) CB/
PDPMA
composition (mole
molecular
weight (g/
gmol) (Mn)
a
fraction) CB/
polydispersity
b
PDPMA
ratio
96/4
96.5/3.5
65 000
1.35
a
1
The composition of the copolymer was determined by H NMR.
The molecular weight and polydispersity ratio of the copolymer were
b
determined by GPC.
1
1
was estimated to be 3.5 mol % by H NMR according to the
proton signals from the pyridyl disulfide at 7.0−8.5 ppm and
the methyl group of the copolymer main chain at 2.5 ppm. The
average molecular weight (Mn) is 65 000 as measured by gel
permeation chromatography (GPC). Other ratios of PDPMA
to CB were prepared to synthesize the copolymer; however, it
was found that the copolymer was not dissolved in water when
the ratio of PDPMA to copolymer was greater than 5 mol %.
Therefore, 3.5 mol % of PDPMA in the copolymer was used in
the study.
under vacuum. Yield: 0.29 g, 74.4%. H NMR (CD3OD): δ 1.90 (s,
−S−C(−CN, −CH2−)−CH3); 3.75 (t, −(CO)−O−CH2−CH2−
O−); 4.28 (t, −(CO)−O−CH2−CH2−O−); 1.32−1.74 (b,
−(CH−CH2)n−); 1.81−2.07 (b, −(CH−CH2)n− and −CH2−
CH2−CH2−); 2.56−2.68 (t, −CH2−COO−); 2.29−3.12 (b, CH3−
N+−CH3); 3.08−3.35 (b, −CH2−CH2−CH2−); 3.51−3.60 (t, N+−
CH2−CH2−C−OO−).
2.4. Preparation of Hydrogels. Disulfide-cross-linked pCB
hydrogels were prepared via thiol−disulfide exchange reaction with
pCB-DT as a cross-linker (Figure 1A). The copolymer, pCB-PDP, was
dissolved in PBS to make 3.5 and 7% (w/v) in a 100 μL solution,
respectively. Three amounts, 0.5, 1, and 2 mg of pCB-DT, were added
into the copolymer solution and allowed to gel. Gelation was observed
by inverting the tubes until the gel solution stopped flowing. PEGMA
hydrogels were prepared following the procedure as illustrated in
Figure S1, Supporting Information.
2.5. Cell Encapsulation in pCB and RGD-pCB Hydrogels.
Three cell types and three cell-packing densities were encapsulated in
the pCB hydrogels and evaluated for cell viability. The three cell types
studied were NIH3T3 fibroblasts, MG63 osteoblast-like cells, and
HepG2 hepatocarcinoma cells; three cell-seeding densities, including 5
× 104, 2 × 105, and 1 × 106 cells/mL, were used. Cells were suspended
in their respective medium and mixed with pCB-PDP and pCB-DT to
allow gelation. Here, the final concentrations of pCB-PDP and pCB-
DT in the cell suspension were 7% and 2% (w/v), respectively.
Similarly, cells were also suspended in pPEGMA-PDP and PEG-DT
for encapsulation as illustrated in Figure S1, Supporting Information.
Hydrogels embedded with cells were incubated at 37 °C in 5% CO2
and 100% relative humidity. After encapsulation, cells were
immediately stained with 1 mg/mL FDA and 1 mg/mL PI for 30
min at 37 °C in the dark. Samples were washed with a large amount of
PBS and then observed under a Nikon Eclipse TE2000-U
fluororescence microscope. Cell viability was examined by Trypan
Blue staining. The cell-laden hydrogels were immersed into a
hydrogel-dissociation medium (cell medium contained with 2 mM L-
cysteine) at 37 °C for 10 min. The cell suspension was then collected
and stained with Trypan Blue. Dead cells showed a dark blue color
under the microscope, while live cells showed a light color. Viability
was calculated as the number of live cells/the number of total cells ×
100(%).
Herein, the thiol-terminated zwitterionic cross-linker was
prepared through two steps. In step 1, the dithioester-
terminated polymer (CTP-pCB-CTP) was prepared via
RAFT polymerization using a specially designed difunctional
chain transfer agent (CTA) (CTP-DEG-CTP). In step 2, the
dithioester end groups were converted to thiols via a well-
known aminolysis reaction.17 The maximum UV absorbance at
318 nm is attributed to the CTP dithioester end groups on
CTP-pCB-CTP. After the dithioester is converted to a thiol, the
UV absorbance at 318 nm is significantly decreased (Figure 2).
Figure 2. UV spectra of CTP-pCB-CTP and pCB-DT (2 × 10−4 mol/
L) in methanol at room temperature. Dithioester end groups in CTP-
pCBAA-CTP show a high signal at 318 nm. After being converted to
thiols, the peak at 318 nm disappears.
A 5 × 105 cells/mL concentration of cell suspension was mixed with
pCB-PDP and cross-linkers to evaluate cell proliferation. For
preparation of RGD-pCB hydrogels, the cross-linkers contained
equal molar pCB-DT and CGRGDSGC. Cell number was quantified
via use of a cell-counting chamber. Cells were collected from
dissociated hydrogels. After mixing with Trypan Blue, the cells were
loaded onto a hemocytometer and then counted under a microscope.
Cell numbers are normalized against the initial feed cell number.
This indicated that the reaction efficiency of converting
dithioester to thiol is very high. For methacrylate polymer
(i.e., pCBMA), when the thiol functionality generates through
aminolysis of a CTP end group, there is a chance of forming a
thiolactone structure via “backbiting” cyclization.17 This
cyclization results in the loss of thiol functionalities, leading
to the consequent failure of hydrogel formation. Therefore,
pCBAA (polycarboxybetaine acrylamide) was chosen to
prepare the thiol-terminated polymeric cross-linker, pCB-DT.
3.2. Preparation of Hydrogels. pCB-based hydrogels
were formed within a short period of time by simply mixing
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of pCB-PDP and
pCB-DT. In order to make an overall zwitterionic hydrogel, we
prepared a thiol-terminated zwitterionic cross-linker that can
react with the pyridyl disulfide-contained polyzwitterion via a
thiol−disulfide exchange reaction. Chemical compositions and
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pCB-PDP copolymer and pCB-DT cross-linker in PBS or cell
medium. The thiol group of pCB-DT was coupled to the
pyridyl disulfide of pCB-PDP via a thiol−disulfide exchange
reaction, releasing pyridine-2-thioe.18 The existent disulfide-
linked hydrogels can be degraded by a biological reducing
agent, such as glutathione (GSH) or cysteine.19 Here, various
feed ratios of pCB-PDP to pCB-DT for making a hydrogel
were tested. The results of CB hydrogel preparation with
various copolymer (pCB-PDP)/cross-linker (pCB-DT) feed
ratios are summarized in Table 2. The two concentrations of
PCB-PDP were fixed at 3.5% and 7% (w/v) solutions,
respectively. Three amounts, 0.5, 1, and 2 mg, corresponding
to 0.5%, 1%, and 2% (w/v) of pCB-DT, were added into the
copolymer solution, respectively. Before mixing with pCB-DT,
the 3.5% and 7% (w/v) copolymer solutions were liquids of
low viscosity. As 0.5, 1, or 2 mg of pCB-DT was added into the
3.5% copolymer solution, all conditions did not lead to a
hydrogel. Therefore, we increased the copolymer concentration
to 7% (w/v). Hydrogels were formed within 1 min after mixing
with 1 and 2 mg of pCB-DT but not 0.5 mg of pCB-DT. The
apparent gelation can be seen by inverting the tubes (Figure
1C). The 7% (w/v) of copolymer and 2% of cross-linker was
chosen to make a hydrogel for cell encapsulation. For
comparison, we also prepared a degradable PEGMA hydrogel
following the same procedure via mixing of pPEGMA-PDP and
PEG-DT (Scheme S1 and Figure S1, Supporting Information).
3.3. Cell Encapsulation in Hydrogels. To demonstrate
the biocompatibility of the pCB materials and the process of
gelation, three cell types, including NIH3T3 cells, MG63 cells,
and HepG2 cells, and three cell-seeding densities, including 5 ×
104, 2 × 105, and 106 cells/mL, were encapsulated in the pCB
hydrogels. pCB-PDP and pCB-DT were dissolved in cell
suspensions and then formed cell encapsulation in the
Table 2. Preparation of pCB Hydrogels
copolymer concentration (pCB-
PDP) (%, w/v)
cross-linker concentration
(pCB-DT) (%, w/v)
gelation
3.5
3.5
3.5
7
0.5
1
no
no
no
no
yes
yes
2
0.5
1
7
7
2
Figure 3. (A) Fluorescent images of NIH3T3, MG63, and HepG2 cells (106 cells/mL) encapsulated in pCB hydrogels. Viable cells were stained with
Live/Dead staining. Green: live cells. Red: dead cells. Scale bar = 100 μm. (B) Viability of NIH3T3, MG63, and HepG2 cells encapsulated in pCB
and PEGMA hydrogel was evaluated for different culture periods. Error bars represent standard deviation from three independent measurements.
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Figure 4. Cell proliferation in the 3D encapsulation of pCB and RGD-pCB hydrogels examined for 1-, 3-, and 5-day culture. Cell numbers are
normalized against the initial feed cell number (5 × 105 cells/mL). Error bars represent standard deviation from 4 independent measurements: (*) p
< 0.05; (***) p < 0.001.
hydrogels. The viability of encapsulated cells in the hydrogels
was observed under a fluorescent microscope by live/dead
staining. Figure 3A shows that all three cell types are well
distributed in the hydrogel, and the cells residing inside the
hydrogels are fully encapsulated. The vast majority of the cells
were viable within the hydrogels after gelation, and up to 95%
of the cells were alive at the initial stage of cell culture (Figure
3B). The results confirmed that the thiol−disulfide exchange
reaction is nontoxic for cells.
Contrasted with high cell-seeding densities, the viability of
L929 cells with a low cell density (1.8 × 104 cells/mL) was
reported to be 92% for 8-day culture in a 2-methacryloylox-
yethyl phosphorylcholine (MPC)-co-n-butyl methacrylate
(BMA)-co-p-vinylphenylboronic acid (VPBA)/polyvinyl alco-
hol (PVA) (PMBV/PVA) hydrogel in the presence of serum in
a microfluidic chip.22 They attributed high cell viability to (a)
the high gas and solution permeability and solute permeability
of methacryloyloxyethyl phosphorylcholine (MPC)-based
hydrogels and (b) the promoted mass transportation of flow
when the cells were cultured in a microfluid chip.22 However,
cells cultured in medium in a 96-well microplate only
maintained a 56.7% viability after 8 days and were entirely
dead after 12 days.22 These MPC-based hydrogels were
composed of MPC copolymer (60% MPC unit, 30% MBA
unit and 10% VPBA unit) and PVA. Here, we designed an
overall zwitterionic hydrogel, pCB hydrogel, and demonstrated
an outstanding viability in the pCB hydrogel without a fluid
system, with low cell-seeding densities and under serum-free
conditions. Cells were encapsulated in an overall zwitterionic
hydrogel to better mimic three-dimensional matrices for cell
growth.
3.4. Cell Encapsulation in RGD-pCB Hydrogels. After
the three cell types were encapsulated in the pCB hydrogels,
the number of cells did not obviously change throughout a 5-
day culture (Figure 4). To improve cell−matrix interactions or
enhance cell proliferation, it is highly desirable that the
hydrogels have cell-specific adhesion. Here, we designed the
RGD-decorated hydrogel to promote cell proliferation by
adding a cystine-terminated RGD as a cross-linker to react with
the copolymer (Figure 2B). We examined the proliferation of
the three cell types in 3D encapsulation after 1-, 3-, and 5-day
cultures. Figure 4 shows that the RGD-pCB hydrogel promoted
proliferation of all cell types as compared to the pCB hydrogel.
Particularly, both NIH3T3 and MG63 cells proliferated
significantly in the RGD-pCB hydrogel after a 5-day culture.
These results confirmed that incorporation of the RGD peptide
The average viability of the three cell types in the pCB
hydrogels was around 90% under serum-free conditions up to
9-day culture (Figure 3B). The 9-day culture of NIH3T3 cells
in the pCB hydrogels with an initial cell-seeding density of 5 ×
104, 2 × 105, and 106 cells/mL achieved 93.2%, 96.2%, and
94.5% viability, respectively. The viabilities of MG63 cells at 9-
day culture were 93.3% (5 × 104 cells/mL), 92.1% (2 × 105
cells/mL), and 92.0% (106 cells/mL), respectively. The
viabilities of HepG2 cells at 9-day culture were 82.7%, 88.9%
,and 91.4% with an initial cell-packing density of 5 × 104, 2 ×
105, and 106 cells/mL, respectively. It is worth mentioning that
cells exhibited up to 90% survival even though a low cell-
seeding density (5 × 104 cells/mL) under serum-free
conditions was used for cell encapsulation within the pCB
hydrogels. We found that cell viability in the PEGMA hydrogel
was better at high cell-seeding densities of 106 cells/mL than at
low cell-seeding densities of 5 × 104 cells/mL (Figure 3B).
However, results for all cell types showed lower viability in the
PEGMA hydrogel than in the pCB hydrogel with all initial cell-
seeding densities. Generally, cell-seeding densities of 106−107
cells/mL are used for cell encapsulation since cell−cell
communication promotes cell survival.20,21 It was reported
previously that a minimum seeding density of 107 cells/mL was
necessary to maintain the survival of encapsulated β-cells in a
PEG hydrogel. Bazou et al. reported that at concentrations
greater than 106 cells/mL HepG2 cells in alginate showed high
viability.21
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dx.doi.org/10.1021/la303390j | Langmuir 2012, 28, 17778−17784

Langmuir
Article
(4) Keefe, A. J.; Jiang, S. Poly(zwitterionic)protein conjugates offer
increased stability without sacrificing binding affinity or bioactivity.
Nat. Chem. 2011, 4, 59−63.
(5) Cao, Z.; Yu, Q.; Xue, H.; Cheng, G.; Jiang, S. Nanoparticles for
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zwitterionic carboxybetaine hydrogels with a carboxybetaine dimetha-
crylate crosslinker. Biomaterials 2011, 32, 961−968.
into the pCB hydrogels promoted cell proliferation. This work
suggests that it is possible to use pCB hydrogels to provide 3D
aqueous environments for tissue regeneration.
The requirements of hydrogels to be used in biomedical
applications include biocompatibility, biodegradability, and
nonimmunogenicity.13 Furthermore, resistance to nonspecific
protein adsorption and cell adhesion is necessary for tissue
scaffolds. In some applications such as islet encapsulation,
nonfouling in immune protection and host reaction is even
more critical.23 pCB hydrogels provide a friendly environment
for cells while also incorporating a specifically binding 3D
environment for tissue engineering with incorporated func-
tional peptides.
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4. CONCLUSIONS
This study demonstrates the potential use of a zwitterionic
hydrogel, pCB hydrogel, as a cell-encapsulating scaffold. The
biodegradable pCB hydrogel was prepared by a thiol−disulfide
exchange reaction with a pyridyl dithiol-containing CB
copolymer (pCB-PDP) and a polyCB dithiol cross-linker
(pCB-DT). The survival of cells encapsulated in the hydrogel
was up to 90% after 9-day culture. Results show that cells
maintained higher viability when encapsulated in the pCB
hydrogels than the PEGMA hydrogels. Bioactive molecules can
be easily incorporated into this pCB hydrogel system. As the
hydrogel was incorporated with cysteine-terminated cross-
linker, the RGD-pCB hydrogel enhanced cell proliferation in
3D hydrogel matrices.
(9) Chien, H. W.; Tsai, W. B.; Jiang, S. Direct cell encapsulation in
biodegradable and functionalizable carboxybetaine hydrogels. Bio-
materials 2012, 33, 5706−5712.
(10) Nicodemus, G. D.; Bryant, S. J. Cell encapsulation in
biodegradable hydrogels for tissue engineering applications. Tissue
Eng., Part B: Rev. 2008, 14, 149−165.
(11) Yu, L.; Ding, J. Injectable hydrogels as unique biomedical
materials. Chem. Soc. Rev. 2008, 37, 1473−1481.
(12) Burdick, J. A.; Anseth, K. S. Photoencapsulation of osteoblasts in
injectable RGD-modified PEG hydrogels for bone tissue engineering.
Biomaterials 2002, 23, 4315−4323.
(13) Zhu, J. Bioactive modification of poly(ethylene glycol)
hydrogels for tissue engineering. Biomaterials 2010, 31, 4639−4656.
(14) Choh, S. Y.; Cross, D.; Wang, C. Facile synthesis and
characterization of disulfide-cross-linked hyaluronic acid hydrogels
for protein delivery and cell encapsulation. Biomacromolecules 2011,
12, 1126−1136.
ASSOCIATED CONTENT
* Supporting Information
■
S
́
(15) Zhang, Z.; Vaisocherova, H.; Cheng, G.; Yang, W.; Xue, H.;
Detailed description of pPEGMA-PDP synthesis and PEGMA
hydrogel preparation. This material is available free of charge
via the Internet at http://pubs.acs.org.
Jiang, S. Nonfouling behavior of polycarboxybetaine-grafted surfaces:
structural and environmental effects. Biomacromolecules 2008, 9,
2686−2692.
(16) Vaisocherova, H.; Yang, W.; Zhang, Z.; Cao, Z.; Cheng, G.;
Piliarik, M.; Homola, J.; Jiang, S. Ultralow fouling and functionalizable
surface chemistry based on a zwitterionic polymer enabling sensitive
and specific protein detection in undiluted blood plasma. Anal. Chem.
2008, 80, 7894−7901.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sjiang@u.washington.edu (S.J.); weibortsai@ntu.edu.
tw (W.-B.T.). Fax: (+01) 206-543-3778 (S.J.); (+886) 2632-
3040 (W.-B.T.).
■
(17) Xu, J.; He, J.; Fan, D.; Wang, X.; Yang, Y. Aminolysis of
Polymers with Thiocarbonylthio Termini Prepared by RAFT
Polymerization: The Difference between Polystyrene and Polymetha-
crylates. Macromolecules 2006, 39, 8616−8624.
Author Contributions
§These authors contributed equally.
(18) Chong, S. F.; Chandrawati, R.; Stadler, B.; Park, J.; Cho, J.;
̈
Notes
Wang, Y.; Jia, Z.; Bulmus, V.; Davis, T. P.; Zelikin, A. N.; Caruso, F.
Stabilization of polymer-hydrogel capsules via thiol-disulfide exchange.
Small 2009, 5, 2601−2610.
The authors declare no competing financial interest.
(19) Meng, F.; Hennink, W. E.; Zhong, Z. Reduction-sensitive
polymers and bioconjugates for biomedical applications. Biomaterials
2009, 30, 2180−2198.
ACKNOWLEDGMENTS
■
This work was supported by the Office of Naval Research
(N000140910137). H.W.C. was also supported by the
Graduate Students Study Abroad Program, National Science
Council (Taiwan) (100-2917-I-002-035).
(20) Lin, C. C.; Anseth, K. S. Cell-cell communication mimicry with
poly(ethylene glycol) hydrogels for enhancing beta-cell function. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 6380−6385.
(21) Bazou, D.; Coakley, W. T.; Hayes, A. J.; Jackson, S. K. Long-
term viability and proliferation of alginate-encapsulated 3-D HepG2
aggregates formed in an ultrasound trap. Toxicol. In Vitro 2008, 22,
1321−1331.
(22) Xu, Y.; Sato, K.; Mawatari, K.; Konno, T.; Jang, K.; Ishihara, K.;
Kitamori, T. A microfluidic hydrogel capable of cell preservation
without perfusion culture under cell-based assay conditions. Adv.
Mater. 2010, 22, 3017−3021.
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