Triterpenoid-Based Self-Healing Supramolecular Polymer Hydrogels Formed by Host–Guest Interactions

Article abstract of DOI:10.1002/chem.201603753

Pentacyclic triterpenoids, a class of naturally bioactive products having multiple functional groups, unique chiral centers, rigid skeletons, and good biocompatibility, are ideal building blocks for fabricating versatile supramolecular structures. In this research, the natural pentacyclic triterpenoid glycyrrhetinic acid (GA) was used as a guest molecule for β-cyclodextrin (β-CD) to form a GA/β-CD (1:1) inclusion complex. By means of GA and β-CD pendant groups in N,N′-dimethylacrylamide copolymers, a supramolecular polymer hydrogel can be physically cross-linked by host–guest interactions between GA and β-CD moieties. Moreover, self-healing of this hydrogel was observed and confirmed by step-strain rheological measurements, whereby the maximum storage modulus occurred at a [GA]/[β-CD] molar ratio of 1:1. Additionally, these polymers displayed outstanding biocompatibility. The introduction of a natural pentacyclic triterpenoid into a hydrogel system not only provides a biocompatible guest–host complementary GA/β-CD pair, but also makes this hydrogel an attractive candidate for tissue engineering.

Full text of DOI:10.1002/chem.201603753

A Journal of
Accepted Article
Title: Triterpenoid-based self-healing supramolecular polymer
hydrogels formed by host-guest interactions
Authors: Ying Li, Jianzuo Li, Xia Zhao, Qiang Yan, Yuxia Gao, Jie Hao,
Yong Ju, and Jun Hu
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To be cited as: Chem. Eur. J. 10.1002/chem.201603753
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Triterpenoid-based self-healing supramolecular polymer
hydrogels formed by host-guest interactions
Ying Li,[a,d]† Jianzuo Li,[a,d]† Xia Zhao,[a] Qiang Yan,*[b] Yuxia Gao,[c] Jie Hao,[c] Jun Hu,*[a] and Yong Ju[c]
Abstract: Pentacyclic triterpenoids, a class of naturally bioactive
products which have multiple functional groups, unique chiral centers,
rigid skeletons, and good biocompatibility, are ideal building blocks for
fabricating versatile supramolecular structures. In this research the
natural pentacyclic triterpenoid, glycyrrhetinic acid (GA), was used as
a novel guest molecule to bind β-cyclodextrin (β-CD), thus forming a
GA/β-CD inclusion complex (1:1). By attaching GA and β-CD pendant
groups in a copolymer with N,N’-dimethylacrylamides, respectively, a
supramolecular polymer hydrogel can be physically cross-linked by
host-guest interactions between GA and β-CD moieties. Moreover,
the self-healing property of this hydrogel was observed and confirmed
by step-strain rheological measurements, where the storage modulus
supramolecular organogel was reported by Bag and co-workers
in 2005,[14] a series of pentacyclic triterpenoid-based small
molecules have been designed and synthesized to study their
supramolecular gelation behaviors.[15-24] For example, Mezzenga
et al reported that the right-handed fibrillar networks in the
hydrogel of glycyrrhizic acid could be utilized as scaffolds for
hybrid nanomaterials in heterogeneous catalysis;[15] our group
found that a pyridinium-tailored glycyrrhetinic acid amphiphile
could transfer and magnify its chirality to supramolecular level,
consequently resulting in the formation of helical nanofibers.[16]
However, these low molecular weight gels have limitations in
applications either due to the participation of toxic organic
was at a maximum with the molar ratio of [GA]/[β-CD] 1:1. Additionally, solvents, or the weak mechanical properties. Thus, it is necessary
these polymers displayed outstanding biocompatibility. The
introduction of natural pentacyclic triterpenoid biocompound in
hydrogel system not only provides a novel biocompatible guest-host
complementary pair of GA/β-CD, but also makes this hydrogel an
attractive candidate for tissue engineering.
to explore pentacyclic triterpenoids-based polymeric hydrogels,
which will may open a new scenario for pentacyclic triterpenoid-
based biomaterials.
Self-healing polymeric hydrogels exhibit improved safety
and extended lifetime in comparison with traditional hydrogels.
Furthermore, they can restore their initial properties after the
interior or exterior cracks attributed to the dynamic/reversible
linkages in the hydrogel networks.[25-28] To date, a variety of self-
healing hydrogels have been designed for diverse applications,
such as biosensors, drug delivery systems, wound healing, and
shape memory materials.[29-36] Nonetheless, reports about using
abundant biocompatible natural products as building blocks in
self-healing hydrogels are still rare.[32] Therefore, designing self-
healing polymeric hydrogels based on natural pentacyclic
triterpenoids, which possess good mechanical strength, structural
stability and biocompatibility, remains a considerable challenge.
One efficient approach is to utilize a noncovalently system
to construct self-healing hydrogels, since they can autonomously
restore their initial properties.[37-39] Among these approaches,
host-guest interactions is crucial as it combines multiple dynamic
interactions and forms dynamic reversible inclusion complex,
playing an important role in constructing self-healing hydrogels.[40-
46] In these works, azobenzene, ferrocene, and adamantyl groups
were utilized as guest molecules attached to different functional
main chains, thus resulting in self-healing hydrogels by the
complexation with β-cyclodextrin (β-CD). In order to explore
biocompatible compounds as guests, herein, glycyrrhetinic acid
(GA, a natural pentacyclic triterpenoid) and β-CD were used as
the novel guest and host molecules, respectively, both of which
are natural biocompatible compounds. They copolymerized with
N,N’-dimethylacrylamide (DMA) to afford the copolymers
Poly(DMA-GA) and Poly(DMA-CD) with different fractions (Figure
1). Due to the dynamic host-guest interactions between GA and
β-CD units, this supramolecular hydrogel exhibited good self-
healing properties. Moreover, these polymers displayed
Introduction
Pentacyclic triterpenoids, as a class of naturally bioactive
products, are abundant in many plants in the form of free acids or
aglycones.[1-5] Generally, they consist of six isoprene units with
multiple functional groups, unique chiral centers, rigid skeletons,
and good biocompatibility. These features not only endow them
pharmacological properties,[6-9] but also render them as ideal
building blocks for self-assembling versatile supramolecular
structures.[10-13] Since the first pentacyclic triterpenoid-based
[a]
Dr. Y. Li, J. Li, Dr. X. Zhao, Dr. J. Hu
State Key Lab of Polymer Physics and Chemistry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences
Changchun 130022, China
E-mail: jhu@ciac.ac.cn
[b]
Dr. Q. Yan
Department of Macromolecular Science, State Key Laboratory of
Molecular Engineering of Polymers, Fudan University, Shanghai
200433, China
Email: yanq@fudan.edu.cn
[c]
[d]
Y. Gao, J. Hao, Dr. Y. Ju
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus
Chemistry & Chemical Biology, Ministry of Education, Tsinghua
University, Beijing 100084, China
Dr. Y. Li, J. Li
College of Chemistry and Material Science, Shandong Agricultural
University, Tai’an 271018, China
†These two authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.
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ratio[a]
100:1
outstanding biocompatibility, making them attractive candidates
for tissue engineering.
Poly(DMA-GA-
1%)
91:1
54:1
26:1
10:1
96
87
92
89
9600
13000
8300
7900
1.95
1.32
1.83
1.93
Poly(DMA-GA-
2%)
50:1
25:1
10:1
Poly(DMA-GA-
4%)
Poly(DMA-NPA)
[a] The molar ratio of DMA to GA/NPA monomer in the feed; [b] the actual
ratio is calculated from 1H NMR integration; [c] yield calculated from the
insoluble portion in diethyl ether; [d] determined by GPC.
The formation and mechanical properties of supramolecular
polymer hydrogels
The typical procedure for hydrogel formation was as follows:
Poly(DMA-GA) and Poly(DMA-CD) were dissolved in DI water,
respectively, and then mixed at different molar ratio between GA
and CD units (3:1, 2:1, 1:1, 1:2, and 1:3) with different polymer
concentrations, consequently resulting in the formation of
hydrogels.
To clarify the network formation induced by inclusion
1
1
complexation of GA and CD moieties, H NMR, 2D H NOESY
NMR, theoretical computation, X-ray diffraction (XRD) and
isothermal titration calorimetry (ITC) were performed. In 1H NMR
spectra of Poly(DMA-GA-2%), no signal of methyl groups on GA
moieties was observable in D2O (Figure 2a, bottom), indicating
the formation of aggregates by sequestering hydrophobic GA
units within the interior and projecting hydrophilic
polydimethylacrylamide toward the exterior. Upon addition of β-
CD, the methyl protons (positions 23 to 28) appeared and shifted
downfield (Figure 2a, up), revealing that the polymer became
more hydrophilic due to the complexation of GA with β-CD. It
should be noted that there was a continuous downfield shift of GA
methyl protons until the ratio [CD]/[GA] reached 3:1 (Figure S4).
It may be caused by the random distribution of GA pendants on
the polymer chain. Thus, access to GA units was restricted, which
limited the formation of complex in comparison to free GA as
guest.[47] Obviously, adding more β-CD could shift the equilibrium
in the direction of complexation, leading to a gradual increase in
chemical shift of methyl groups of GA.
Figure 1. Preparation and self-healing process of supramolecular hydrogels
cross-linked by Poly(DMA-GA) and Poly(DMA-CD) based on host-guest
interactions between GA and β-CD units.
Results and Discussion
Polymer design and characteristics
Statistical copolymers consisting of GA or β-CD pendants were
synthesized via a free radical polymerization (Figure 1 and
Scheme S1). The copolymerization of DMA with GA-based
methacrylate provided a water-soluble copolymer Poly(DMA-GA)
bearing 1-4% GA pendant groups. A GA moiety was linked with a
short spacer by an esterification reaction, resulting in a flexible
GA moiety to interact with a host molecule. The characteristic
methyl proton peaks of GA moieties at 0.73, 0.92, 1.05, 1.06, 1.07
and 1.32 ppm were observed clearly, and the molar fractions of
GA units in copolymers were calculated from protons integration
in 1H NMR spectrum (Figure S1). For Poly(DMA-CD), a precursor
polymer Poly(DMA-NPA) was obtained via the copolymerization
of DMA with an active ester monomer (NPA). After that, amino-
CD substituted NPA moieties on Poly(DMA-NPA), resulting in
Poly(DMA-CD). The extent of CD substitution was estimated to
be ca. 93% based on 1H NMR integration ratio of aromatic protons
of NPA moieties versus backbone protons at 1.0-2.0 ppm (Figure
S2). Moreover, FTIR spectra of Poly(DMA-NPA), Poly(DMA-CD),
and amino-CD showed that the peak at 1758 cm-1 decreased
sharply after the amino-CD substitution (Figure S3), further
indicating most of active esters (NPA moiety) were substituted,
consistent with 1H NMR results shown in Figure S2. The
characteristics of these copolymers were summarized in Table 1.
In addition to 1D 1H NMR, cross correlation peaks between
1
GA methyl protons and β-CD protons were observed in 2D H
NOESY NMR spectrum (Figure S5), indicating the close contact
of these protons (the detectable limit of NOE signal < 5 Å). Since
the hydrophobic GA moieties and the hydrophilic β-CD protons
are phase-separated in the aqueous environment, these cross
correlation peaks should be generated from GA and β-CD units in
an inclusion complex.[48] It could be further explained by
theoretical computation. The structure of GA was optimized by
employing B3LYP/6-31G(d,p) basis set in the gas phase with
Gaussian 09 suite of programs. As shown in Figure 2c, the
molecular breadth of GA was 0.58-0.60 nm, which was smaller
than the diameter of β-CD cavity (0.60-0.65 nm). Such size match
enabled GA skeleton to enter into the cavity of β-CD.[49] Moreover,
due to the molecular length of GA (1.27 nm) was larger than the
Table 1. Characteristics of Poly(DMA-GA) and Poly(DMA-NPA)
polymers
feed
ratio[b]
yield%[c]
Mn[d]
Mw/Mn[d]
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height of β-CD (0.79 nm), only partial GA skeleton could be in the
cavity of β-CD when the host-guest complexation formed, which
was consistent with 1H NMR titration results. Apparently, the
synergistic effect of size match, hydrophobic interactions, and van
der Waals forces between GA and β-CD units primarily promoted
the dynamic host-guest complexation, consequently resulting in
the formation of supramolecular polymer hydrogel.
Oscillatory rheology was used to measure mechanical
properties of the hydrogels. As shown in Figure 3a, the highest G’
was obtained when Poly(DMA-GA-2%) and Poly(DMA-CD) were
mixed at a molar ratio of [GA]/[CD] = 1.0 (corresponding to a
weight ratio of 55/22 for the two polymers), ascribing to the
highest cross-linking density at 1:1. When either GA or CD units
were present in excess, a decrease in G’ was observed, indicating
the effective formation of complementary inclusion complex,
which was consistent with the phenomenon in cholic acid and β-
CD complexation hydrogel system.[32] Meanwhile, an increase in
concentration of polymers led to an increasing G’ at [GA]/[CD] =
1.0, consequently affording a tougher hydrogel as shown in Figure
3b. Moreover, lower G’ values were obtained with the decrease in
GA fraction of Poly(DMA-GA) at the same polymer concentration
(Figure 3c), demonstrating the effect of cross-linking density on
the strength of hydrogels. Based on the above results, it is
apparent that the mechanical property of the complex hydrogel is
greatly influenced by the molar ratio of [GA]/[CD], concentration
of polymers, and GA fraction of Poly(DMA-GA), which primarily
determine the cross-linking density of these two components.
Figure 3d showed the native morphology of Poly(DMA-GA-
2%)/Poly(DMA-CD) hydrogel at a concentration of 15 wt%. The
hydrogel exhibited porous and interconnected structures with the
pore diameter around 40 μm, offering a comfortable environment
for mass delivery and cell growth in tissue engineering.
More evidence for the complexation of GA and CD moieties
came from XRD results. As shown in Figure S6, Poly(DMA-GA-
2%) was found to be amorphous, and a certain degree of
crystallinity of Poly(DMA-CD) was demonstrated by two broad
crystalline peaks at 2θ = 11.6 and 18.1° in the diffractogram.
Conversely, the diffractogram of Poly(DMA-GA-2%)/Poly(DMA-
CD) hydrogel ([GA]/[CD] = 1:1, 15 wt%) did not show the presence
of peak at 2θ = 18.1°, whereas a broad diffraction peak range of
7-30° was detected. It revealed that the host-guest complexation
between Poly(DMA-GA-2%) and Poly(DMA-CD) was formed.[30]
The binding affinity between GA and β-CD moieties was
determined in ITC experiment. GANa and β-CD were used as the
model guest and host molecules, respectively. As shown in Figure
2b, a typical titration curve of 1:1 complex formation was observed
with the stoichiometric ratio “N” 0.96 from the curve-fitting results.
Moreover, the binding constant was determined as Ka = 1.59 ×
103 M-1, as well as thermodynamic parameters ΔH = -21.10 kJ/mol
and ΔS = -9.47 J/mol.K-1. Thermodynamically, the binding of β-
CD with GANa was entirely driven by the favorable enthalpic
changes accompanied by the unfavorable entropic changes (ΔH°
< 0; TΔS° < 0).[50]
Figure 3. Storage modulus (G’) and loss modulus (G’’) (time sweep at 25 °C) of
Poly(DMA-GA-2%)/Poly(DMA-CD) hydrogel in relationship to (a) the molar ratio
of GA to CD units (15 wt %), and (b) the concentration of the polymer complex
([CD]/[GA] = 1); (c) storage modulus (G’) and loss modulus (G’’) (frequency
sweep at 25 °C, 15 wt%, [CD]/[GA] = 1) of Poly(DMA-GA-1%)/Poly(DMA-CD),
Poly(DMA-GA-2%)/Poly(DMA-CD), and Poly(DMA-GA-4%)/Poly(DMA-CD); (d)
SEM image of Poly(DMA-GA-2%)/Poly(DMA-CD) hydrogel (15 wt%, [CD]/[GA]
= 1).
Self-healing ability
Figure 2. (a) 1H NMR spectra of Poly(DMA-GA-2%) in D2O (bottom) and
Poly(DMA-GA-2%) mixed with β-CD in D2O (25 oC, conc. 10 mg/mL, [CD]/[GA]
= 3:1) (up); (b) Calorimetric titration curve of GANa with β-CD in DI water at
25 °C. Raw data for sequential 10 μL injections of solution β-CD (4.48 mM) into
solution GANa (0.25 mM) (up); Heats of reactions as obtained from the
integration of the calorimetric traces (bottom); (c) Theoretical optimized
structure of GA. Carbon atoms, hydrogen atoms, and oxygen atoms are
presented in gray, light gray, and red, respectively.
To demonstrate the self-healing behavior of Poly(DMA-
GA)/Poly(DMA-CD) hydrogels at room temperature, two
cylindrical hydrogels stained with erioglaucine disodium salt
(EDS) and tartrazine (TAR) (Figure 4a), respectively, were cut into
two pieces (Figure 4b) and then put together. After 60 s, the two
pieces of hydrogel healed (Figure 4c). Optical microscopy image
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showed that EDS and TAR which were used to stain hydrogels
interpenetrated quickly with each other resulting in the green color
(Figure 4d). The above results showed that the hydrogels
possessed good self-healable ability as well as appropriate
permeation for bioactive agent delivery.
Cell viability
Fibroblast 3T3-L1 cells were co-cultured with polymers to
investigate their cytotoxicity. On account of the natural origin of
GA and β-CD, these polymers displayed outstanding
biocompatibility even with the concentrations up to 5.0 mg/mL
using CellTiter-BlueTM cell viability assay (Promega) (Figure 5a).
As shown in Figure 5b-c, live cells were stained green and dead
cells were in red color, indicating that the majority of 3T3-L1 cells
survived in presence of these polymers (5 mg/mL) for 24 h.
However, cell viability and morphology were the only parameters
examined in this study as preliminary indicators of the suitability
of Poly(DMA-GA)/Poly(DMA-CD) hydrogel for tissue engineering
applications. Future studies will include the construction of cells-
contained self-healing multilayer structures to join different
materials in hybrid tissue engineering, aiming to overcome the
disadvantages in suturing and gluing methods at present, such as
the formation of voids, the introduction of adhesive materials
between discrete layer compartments.
Rheology measurements were performed to confirm the
self-healing ability of hydrogels. The strain amplitude sweep was
employed to monitor G’ and G’’ of Poly(DMA-GA-2%)/Poly(DMA-
CD) hydrogel. As shown in Figure 4e, under the strain from 0.1%
to 80%, G’ was larger than G’’, suggesting that the hydrogel
networks were stiff and the cross-linkage remained undamaged
under relatively large deformations. With G’ and G’’ curves
crossed, a gel-sol transition point occurred at the strain of 110%,
which implied the beginning of hydrogel networks destruction due
to the disruption of GA-CD interaction at such strain.[39] It was
shown that G’ was smaller than G’’, and G’ dramatically
decreased to 63 Pa at the large strain of 650%, suggesting the
cross-linking networks were completely collapsed. According to
the results of strain sweep, the self-repairing capacity of
Poly(DMA-GA-2%)/Poly(DMA-CD) hydrogels was verified by the
alternate step strain test (strain = 3 and 250%) as shown in Figure
4f. By switching the strain from a large strain of 250% to a small
strain of 3% at a fixed angular frequency (10 rad/s), G’ and G’’
recovered to the initial data without the significant decrease in
each cycle of the recovery. The above studies showed that
Poly(DMA-GA-2%)/Poly(DMA-CD) hydrogels recovered their
original properties rapidly after being sheared. Therefore, the self-
healing mechanism for this supramolecular hydrogel was inferred
to be the reversible host-guest inclusion complexation between
GA and CD units. When the host-guest complexes disassembled,
the hydrogel was broken. At rest, the complexes formed again,
leading to a self-healing behavior.[51] It was worth noting that the
self-healing behavior of the supramolecular polymer hydrogel
occurred autonomously without any external intervention, which
was advantageous in comparison to the gels that need external
treatment.
Figure 5. Cell viability (a) and cell images after treatment with Poly(DMA-CD)
(b), Poly(DMA-GA-1%) (c), Poly(DMA-GA-2%) (d), and Poly(DMA-GA-4%) (e)
for 24 h. Cells were stained with FDA (green color) and PI (red color).
Conclusions
In summary, we utilized a natural biotriterpenoid, glycyrrhetinic
acid (GA), as a novel guest molecule to bind β-CD (1:1) for the
first time, thus forming the GA/β-CD inclusion complex. Relying
on this, a supramolecular polymer hydrogel was formed by mixing
N,N’-dimethylacrylamides copolymers which attached GA and β-
CD pendants, respectively. Our results showed the storage
modulus was highest when the molar ratio of [GA]/[β-CD] was 1:1.
Moreover, such supramolecular hydrogel exhibited good self-
healing property, which not only can be seen visually, but also
confirmed by step-strain rheological measurements. Additionally,
these polymers displayed outstanding biocompatibility. This work
not only provides a novel guest/host complex GA/β-CD, but also
makes the hydrogel an attractive candidate in tissue engineering.
Figure 4. Photographs of the self-healing process: (a) two cylindrical hydrogels
of Poly(DMA-GA-2%)/Poly(DMA-CD) (30 wt%, [CD]/[GA] = 1; left: EDS, right:
TAR); (b) hydrogels were cut in half; (c) the self-healed hydrogel; (d) optical
microscopy image of the healed hydrogel; Rheological characterization of self-
healing hydrogels: (e) G’ and G’’ of Poly(DMA-GA-2%)/Poly(DMA-CD) (7 wt %,
[CD]/[GA] = 1) as a function of strain (0.1 - 650%), and (f) G’ and G’’ of hydrogels
in alternate step strain measurements (25 oC). Large strain (250%) inverted the
values G’ and G’’ to give the sol state. On the other hand, G’ was recovered
under a small strain (3%).
Experimental Section
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500-600 nm fluorescence, while PI was excited with 555 nm laser, emitting
560-700 nm fluorescence.
Materials
Acryloyl chloride, methacryoyl chloride, β-cyclodextrin (β-CD), p-
nitrophenol, p-toluenesulfonyl chloride, ethylenediamine, glycyrrhetinic
acid (GA), erioglaucine disodium salt (EDS), tartrazine (TAR), and other
reagents were local commercial products and used as received. 2,2-
Azoisobutyronitrile (AIBN) was recrystallized twice from ethanol. N,N’-
Dimethylacrylamide (DMA) was distilled before use. Amino-CD, p-
nitrophenyl acrylate (NPA), and glycyrrhetic acid sodium salt (GANa) were
synthesized according to literatures (Scheme S1).[52-54]
Synthesis of GA-based hexanol (GAH)
GA (5.00 g, 10.62 mmol), 6-bromo-1-hexanol (1.67 mL, 12.75 mmol), and
K2CO3 (1.75 g, 12.75 mmol) were dissolved in dry DMF (30 mL), and then
the mixture was stirred at room temperature for 4 h. The reaction was
quenched by diluted hydrochloric acid (N = 1), and extracted by ethyl
acetate. The organic layer was washed by water, brine, and dried by
anhydrous Na2SO4. After removing the solvent under reduced pressure,
the crude was obtained, which was further purified by chromatography
(hexane/ethyl acetate = 2:1, v/v) to afford GAH as a white powder (5.60 g,
93%). ESI-MS (+): m/z 571 [M+H]+; 1H NMR (400 MHz, CDCl3, ppm): δ
5.63 (s, H, 12-CH=C), 4.10 (m, 2H, CO2CH2), 3.64 (t, 2H, J = 6.48 Hz,
HOCH2), 3.21 (m, 1H, 3-H), 1.36, 1.14, 1.11, 0.99, 0.80, 0.79 (s, 7 × 3H, 7
× CH3); 13C NMR (100 MHz, CDCl3, ppm): δ 200.76 (O=C-CH=C), 176.65
(CO2CH2), 170.00 (O=C-CH=C), 128.40 (O=C-CH=C), 78.87, 64.58, 62.66,
62.40, 55.07, 48.73, 45.59, 44.15, 43.41, 41.22, 39.26, 37.81, 37.24, 32.90,
32.81, 31.99, 31.25, 28.95, 28.72, 28.63, 28.23, 27.43, 26.54, 26.23, 25.54,
23.52, 18.82, 17.60, 16.49, 15.71.
Methods
The molecular weight and polydispersity index (PDI) of the polymers were
determined by gel permeation chromatography (GPC), and measured on
a Waters system equipped with HPLC pump (Waters 1515) and a
refractive index detector (Waters 2414). THF was used as the eluent at a
flow rate of 1.0 mL/min at 30 oC, and polystyrene standards were employed
for the GPC calibration. 1H and 13C NMR experiments were carried out in
CDCl3 or D2O on a Bruker AVANCE III HD 400 spectrometer at 25 oC. 2D
1H nuclear Overhauser effect spectroscopy (NOESY) was recorded on
JOEL JNM-ECA 400 apparatus in D2O. FTIR spectra were obtained with
a KBr pellet method between 4000 and 500 cm-1 using VERTEX 70
spectrometer. Isothermal titration calorimetry (ITC) experiment was
performed on a NANO ITC-SVH from TA instrument. Solutions were
degassed and thermostatted using a TA Vac accessory before each
titration. A water solution of β-CD in a 250 μL syringe was sequentially
injected into the calorimeter sample cell (1.42 mL) containing an aqueous
of GANa with a stirring speed at 250 rpm. Each titration experiment was
composed of 25 successive injections (10 μL per injection). X-ray
diffraction (XRD) were conducted on a D8 Discover diffractometer from
Bruker using CuK α radiation. The region of the scanning was recorded
from 2 to 45° with a 2°/min scanning rate. Rheological experiments were
performed on an AR 2000 rheometer from TA Instruments with 25 mm
diameter parallel plates. Rheological gel characteristics were monitored by
oscillatory time sweep, frequency sweep, and strain sweep experiments.
During time sweep experiments G’ (storage modulus) and G’’ (loss
modulus) were measured at 25 oC for a period of 5 min, with a constant
strain of 3 % and frequency of 10 rad/s. Quantum chemistry computations
were performed using the B3LYP density functional theory (DFT) with
Gaussian09 suite of programs. The structure was optimized by employing
the 6-31G(d,p) basis set in the gas phase.
Synthesis of GA-based methacrylate (GAM)
Methacryoyl chloride (340 μL, 3.50 mmol) was added dropwise to a dry
DCM solution of GAH (2.00 g, 3.50 mmol) and triethylamine (583 μL, 4.21
mmol) at 0 oC. After the addition, the reaction was stirred at room
temperature for 5 h. The mixture was quenched by diluted hydrochloric
acid (N = 1), and then extracted by ethyl acetate. The organic layer was
further washed by water, brine, and dried by anhydrous Na2SO4. After
removing the solvent under reduced pressure, the crude was obtained,
which was further purified by chromatography (hexane/ethyl acetate = 6:1,
v/v) to give GAM as a white powder (1.96 g, 88%). ESI-MS (+): m/z 639
[M+H]+; 1H NMR (400 MHz, CDCl3, ppm): δ 6.06 (m, 1H, C=CH2), 5.61 (s,
H, 12-CH=C), 5.52 (m, 1H, C=CH2), 4.10 (t, 2H, J = 6.56 Hz, CO2CH2),
4.06 (t, 2H, J = 6.56 Hz, CO2CH2), 3.21 (m, 1H, 3-H), 1.91, 1.34, 1.11, 1.10,
1.09, 0.97, 0.77 (s, 8 × 3H, 8 × CH3); 13C NMR (100 MHz, CDCl3, ppm): δ
200.24 (O=C-CH=C), 176.54 (CO2CH2), 169.31 (O=C-CH=C), 167.58
(O2C-CH=CH2), 136.54 (O2C-CH=CH2), 128.59 (O=C-CH=C), 125.34
(O2C-CH=CH2), 78.80, 64.68, 64.41, 61.89, 55.02, 44.07, 43.29, 41.16,
39.22, 37.84, 37.16, 32.84, 31.91, 31.20, 28.71, 28.65, 28.61, 28.19, 27.37,
26.56, 26.49, 25.79, 25.75, 23.49, 18.76, 18.43, 17.57, 16.45, 15.69.
Cytotoxicity measurement
Synthesis of Poly(DMA-GA-2%)
Fibroblast cell line 3T3-L1 was provided by Norman Bethune Health
Science Center of Jilin University and grown in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) in an atmosphere of 5 % CO2 at 37 °C. The cells were seeded in
96-well culture plate with a density of 1 × 105 cells per well. After overnight
incubation, the previous media was replaced by fresh DMEM containing
Poly(DMA-GA) and Poly(DMA-CD) at certain concentrations, respectively,
and incubated for 24 h. After that, 20 μL CellTiter-BlueTM reagent was
added to each well and incubated for further 3 h to perform the cell viability
assay. The fluorescence intensity was measured at 560/590 nm (Ex/Em)
using a BioTek Synergy H1 microplate reader.
A mixture of DMA (2.08 g, 20.99 mmol), GAM (0.27 g, 0.42 mmol), and
AIBN (20 mg, 0.12 mmol) were dissolved in dry DMF (5 mL), and then the
mixture was frozen in liquid N2. After removing the system air under
vacuum, the mixture was purged with N2 prior to its immersion in a
preheated oil bath at 70 °C. The reaction was allowed to proceed for 18 h
at 70 °C before being quenched by immersion into ice-water. DMF was
removed under reduced pressure, and THF was added to re-dissolve the
copolymer. After pouring the reaction mixture into diethyl ether, the
precipitate was collected. The copolymer was dried in vacuum to yield
Poly(DMA-GA-2%) (2.18 g, 94%). Poly(DMA-GA-1%) and Poly(DMA-GA-
4%) with different feed ratio between GA and DMA units were synthesized
similar as Poly(DMA-GA-2%).
Cell imaging
The 3T3-L1 cells were seeded in 12-well culture plate with a seeding
density of 4 × 104 cells per well. After overnight incubation, the cells were
treated with DMEM containing Poly(DMA-GA) and Poly(DMA-CD) at the
concentration of 5 mg/mL, and then co-cultured at 37 °C. After 24 h, the
samples were stained with 5 μg/mL FDA and 10 μg/mL PI solution. Cells
were imaged by a LSM 700 confocal laser scanning microscope imaging
system (Carl Zeiss). The FDA was excited using a 488 nm laser, emitting
Synthesis of Poly(DMA-NPA)
DMA (1.70 mL, 16.48 mmol), NPA (0.32 g, 1.64 mmol), and AIBN (29 mg,
0.16 mmol) were dissolved in dry DMF (15 mL), and then the mixture was
frozen in liquid N2. After removing the system air under vacuum, the
mixture was purged with N2 prior to its immersion in a preheated oil bath
70 °C. The reaction was allowed to proceed for 18 h at 70 °C before being
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10.1002/chem.201603753
Chemistry - A European Journal
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quenched by immersion into ice-water. Copolymer was precipitated from
diethyl ether after removing most DMF, and Poly(DMA-NPA) (1.73 g, 89%)
was obtained after drying in vacuum.
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This work is supported by NSFC (No. 21604085, 21472108),
NBRP of China (973 Program, No. 2012CB821600), Jilin Science
Foundation for Youths (No. 20160520135JH), and Open
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Entry for the Table of Contents (Please choose one layout)
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Glycyrrhetinic acid (GA) was used as a
novel guest molecule to bind β-
cyclodextrin (β-CD), thus forming a
GA/β-CD inclusion complex (1:1). By
attaching GA and β-CD pendant
groups in a copolymer with N,N’-
dimethylacrylamides, respectively, a
self-healing supramolecular polymeric
hydrogel can be physically cross-linked
by host-guest interactions between GA
and β-CD moieties.
Ying Li, Jianzuo Li, Xia Zhao, Qiang
Yan,* Yuxia Gao, Jie Hao, Jun Hu,*
Yong Ju
Page 1. – Page 6.
Triterpenoid-based self-healing
supramolecular polymer hydrogels
formed by host-guest interaction
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