Biological self-assembly of injectable hydrogel as cell scaffold via specific nucleobase pairing

Article abstract of DOI:10.1039/c2cc35449g

A biological hydrogel was self-assembled via Watson-Crick base pairing of thymine and adenine from functionalized star poly(ethylene glycol). Our work should provide a novel methodology to generate robust injectable scaffolds with tailorable properties for biomedical applications.

Full text of DOI:10.1039/c2cc35449g

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Biological self-assembly of injectable hydrogel as cell scaffold via specific
nucleobase pairingw
a
a
a
a
b
Huaping Tan,* Chao Xiao, Jinchen Sun, Dangsheng Xiong and Xiaohong Hu
Received 27th July 2012, Accepted 30th August 2012
DOI: 10.1039/c2cc35449g
A biological hydrogel was self-assembled via Watson–Crick
base pairing of thymine and adenine from functionalized star
poly(ethylene glycol). Our work should provide a novel method-
ology to generate robust injectable scaffolds with tailorable
properties for biomedical applications.
of linear PEG in this study. Firstly, thiol thymine (T-SH) and
thiol adenine (A-SH) were synthesized. Maleimide terminated
four-arm PEG (PEG-Mal) was functionalized with either
T-SH or A-SH functionalities as self-assembly precursors
(referred as PEG-T and PEG-A) via Michael-type addition
(
Fig. 1a and b). After dissolution and mixing of precursors in
an aqueous environment, a stereo-PEG hydrogel network was
self-assembled due to the formation of pairing complexes
(Fig. 1c).
Biodegradable hydrogels have served as highly functional cell
scaffolds for tissue engineering and in delivery of cell growth
1
factors (GFs). Conventionally, biodegradable hydrogels have
0
The storage moduli G of self-assembled hydrogels (PEG
been synthesized by chemical crosslinking methods using cross-
linking agents, photopolymerization, click chemistry and covalent
2
conjugation. While the versatility of crosslinking has been
20 kDa, 20 wt%) were monitored as a function of molar ratio of
PEG-T/PEG-A at 37 1C in PBS (Fig. 2a). When PEG-T/PEG-A
was mixed with different molar ratios, storage moduli were
broadly exploited, chemical hydrogels are limited by irreversible
modification and aggregation of the entrapped proteins and the
potential toxicities of chemically reactive moieties. A major issue
in the use of hydrogels and tissue reconstruction procedures in
general is that the establishment of a biomimic environment into
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the engineered tissue is necessary for engineered tissue survival.
A goal is the synthesis of a biological hydrogel via self-assembly
that might therefore permit manipulation of inductive properties
for cells proliferation. Recently, self-assembly conjugation in
polymeric materials has become increasingly prominent in the
4
production of bioactive surfaces and matrices. A great variety of
self-assembly methods have been reported for conjugation of
peptides, proteins, DNA, oligonucleotides and nanoparticles,
which are based on electrostatic interactions, inclusion complex,
ionic interactions, hydrophobic interactions, hydrogen bonding,
5
crystallinity and affinity interactions.
Here, we present a flexible way to self-assemble a polymeric
hydrogel via specific pairing of functionalized nucleobases.
The biological self-assembly of a poly(ethylene glycol) (PEG)
hydrogel system is established by Watson–Crick base pairing
between thymine (T) and adenine (A) via hydrogen bonding
(
Fig. 1). Compared with linear PEG, multi-arm star-shaped
PEG has the tendency to induce stereo-complex formation, which
6
shows promise in biomedically-relevant hydrogel systems.
Four-arm PEG was herein chosen for self-assembly instead
a
School of Materials Science and Engineering,
Fig. 1 (a, b) Four-arm poly(ethylene glycol)-maleimide (PEG-Mal)
was functionalized with thiol thymine (T-SH) and thiol adenine (A-SH),
respectively, as self-assembly precursors via Michael-type addition.
Nanjing University of Science and Technology, Nanjing 210094,
China. E-mail: hptan@njust.edu.cn
School of Material Engineering, Jinling Institute of Technology,
b
(c) Schematic of biological self-assembly of ideal four-arm PEG hydrogel
Nanjing 211169, China
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c2cc35449g
network via intermolecular hydrogen bonding of Watson–Crick base
pairs between thymine and adenine functionalities.
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10289–10291 10289

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0
G gradually decreased, which was associated with a con-
0
0
0
00
comitant decrease of G . On heating the hydrogel with G 4 G ,
a transition from gel-to-sol occurred at 72 1C, its gel transition
0
0
0
temperature. Above 72 1C, G starts to exceed G , indicating
that the mixtures behave as weak viscous liquids.
As shown in Fig. 3a, the incubation temperature has a
significant influence on weight loss of the self-assembled
hydrogels in PBS. At lower temperatures, the hydrogel showed
a slower weight loss rate than that at higher temperatures. At
5
0 1C, the hydrogel showed a significantly faster weight loss,
due to destruction of hydrogen bonding, and totally dissolved
in 9 days. Compared to the result at 50 1C, the hydrogels lost
weight steadily up to 18 and 24 days at 37 and 20 1C,
respectively. The swelling kinetics of the hydrogel were also
investigated in PBS at different temperature as a function of
incubation time (Fig. 3b). The hydrogel at a lower temperature
showed a lower swelling ratio rate than that at higher tem-
perature, which was consistent with the weight loss data
shown in Fig. 3a. Compared to the 20 and 37 1C data, the
swelling ratio of the hydrogel significantly increased after 24 h
incubation at 50 1C. At 37 1C, the swelling ratio of the
hydrogel significantly increased from 17.3 to 26.8 after incu-
bation for 14 days.
Fig. 2 (a) Storage modulus of self-assembled hydrogels as a function
of PEG-T/PEG-A molar ratio. (b) Storage modulus of self-assembled
hydrogels with different molecular weights and concentrations.
(
c) Rheological characterization of self-assembled hydrogel networks.
The inset image illustrates the formed PEG-20K/20K hydrogel in PBS.
d) Rheological properties of the self-assembled hydrogel as a function
(
of temperature.
GFs that induce adipose-derived stem cells (ASCs) prolif-
eration include insulin, dexamethasone (Dex) and vascular
measured between 63 and 600 Pa. Results indicated that the
storage modulus of the 5/5 hydrogel with equimolar amounts
of PEG-T/PEG-A was the highest (599.4 Pa), which showed a
7
endothelial growth factor (VEGF). Fig. 3c shows the
initial concentration of insulin, dexamethasone and VEGF
entrapped within the hydrogel. The hydrogel showed the
highest efficiency loading of VEGF, and about 96% of VEGF
could be entrapped within the hydrogel, but there was no
significant difference compared with dexamethasone and
insulin (p 4 0.05). The in vitro cumulative amount of GFs
released from the hydrogel at specific time points was quanti-
fied. As observed in Fig. 3d, the GFs incubated in PBS
demonstrated a total cumulative release of approximately
68–89% over the 14-day time period, with total release in
18–20 days. The release of dexamethasone showed a signifi-
cantly faster rate than both insulin and VEGF (p o 0.05).
0
significantly higher G than the others (p o 0.05). Less PEG-T
or PEG-A in mixtures may decrease the number of ligand
units available for hydrogen bonding, which leads to imperfect
networks and lower storage moduli.
Two different molecular weights (10 and 20 kDa) of four-
arm PEG were studied for self-assembly in aqueous solution at
3
7 1C (Fig. 2b). Three self-assembly hydrogels consisting of
PEG-T/PEG-A with molecular weights of 10 or 20 kDa,
e.g. PEG-10K/10K, PEG-10K/20K and PEG-20K/20K were
prepared. The PEG-20K/20K hydrogel showed the highest storage
modulus, followed by PEG-10K/20K and PEG-10K/10K
0
hydrogels. Fig. 2b also showed that G significantly increased
with increasing the polymer concentrations from 10 to 20 wt/wt%.
This indicated that higher concentrations of polymer created a
higher effective cross-link density and therefore led to a tighter
network. Since PEG-20K/20K (20 wt/wt%) comprised more
polymer chains and gave the strongest storage modulus, a
concentration of 20 wt/wt% and molecular weight of 20 kDa
were used for subsequent studies, and further discussion refers
only to this hydrogel.
Dynamic time sweep rheological experiments were con-
ducted to monitor the network evolution and evaluate the
viscoelastic behavior of the hydrogel (Fig. 2c). Upon mixing of
homogeneous PEG-T and PEG-A solutions (20 wt/wt%) at
0
3
7 1C, the storage modulus G was significantly increased and
0
0
larger than the loss modulus G after 150 s. The data indicate a
0
final storage modulus G value of B600 Pa after 500 s, in
0
0
excess of the loss moduli G (o), which resulted in a hydrogel
with a typical viscoelastic property. The inset in Fig. 2c
illustrates the viscoelastic hydrogel after self-assembly in PBS.
Fig. 2d shows the temperature-dependent rheological
characteristics of the hydrogel. With increasing temperature,
Fig. 3 (a) Degradation of the hydrogel at different temperatures in
PBS. (b) Swelling kinetics of the hydrogel at different temperatures.
(c) Entrapped GFs in hydrogel in PBS. (d) Cumulative release profiles
of GFs from hydrogel.
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0290 Chem. Commun., 2012, 48, 10289–10291
This journal is c The Royal Society of Chemistry 2012

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the biological hydrogel was able to preserve the proli-
feration of the encapsulated ASCs. Coupled with the cell
attachment data, our results suggested a novel potential
mechanism for targeted proliferation of stem cells via biological
self-assembly.
As presented, we have developed a novel methodology to
self-assemble a biological four-arm star-shaped PEG hydrogel
as an injectable scaffold that specifically allows for targeted
GFs delivery and cell encapsulation. The formation of the
self-assembled hydrogel is attributed to Watson–Crick base
pairing via intermolecular hydrogen bonding between thymine
and adenine. The potential application of this biological
hydrogel as a cell scaffold in soft tissue engineering was
confirmed by encapsulation behavior of human ASCs. These
unique characteristics of this biological hydrogel make it a
promising candidate as an injectable scaffold for pharma-
ceutical and biomedical applications.
This study is financially supported by the National Natural
Science Foundation of China (51103071), Natural Science
Foundation of Jiangsu Province (BK2011714), Fundamental
Research Funds for the Central Universities Research
(
NUST2011ZDJH12) and Zijin Star program of NJUST.
Notes and references
Fig. 4 (a) Number of adhered ASCs to the surface of hydrogels.
b) Proliferation of ASCs cultured in hydrogels. (c, d) Confocal laser
1
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This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10289–10291 10291