Novel anisotropic supramolecular hydrogel with high stability over a wide pH range

Article abstract of DOI:10.1021/la103982e

The hydrolysis of the carboxylic ester bond, by a base or catalyzed by an enzyme under weak basic conditions, serves as the only path to obtain a novel anisotropic supramolecular hydrogel that is stable over a wide pH range. This result not only expands the molecular scope of supramolecular hydrogelators but also illustrates the design principles for creating pH-stable supramolecular soft materials.

Full text of DOI:10.1021/la103982e

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© 2010 American Chemical Society
Novel Anisotropic Supramolecular Hydrogel with High Stability over
a Wide pH Range†
Fan Zhao,‡,§ Yuan Gao,‡ Junfeng Shi,‡ Hayley M. Browdy,‡ and Bing Xu*,‡
‡Department of Chemistry and §Quantitative Biology Program, Brandeis University, 415 South Street,
Waltham, Massachusetts 02454, United States
Received October 4, 2010. Revised Manuscript Received November 4, 2010
The hydrolysis of the carboxylic ester bond, by a base or catalyzed by an enzyme under weak basic conditions, serves
as the only path to obtain a novel anisotropic supramolecular hydrogel that is stable over a wide pH range. This result
not only expands the molecular scope of supramolecular hydrogelators but also illustrates the design principles for
creating pH-stable supramolecular soft materials.
Supramolecular hydrogels, resulting from the self-assembly of
certain small organic molecules (i.e., hydrogelators) driven by
noncovalent interactions, have emerged as a type of versatile soft
material and have found applications in many areas.1 Because of
their inherent and excellent biocompatibility and biodegradabil-
ity, supramolecular hydrogels have shown promise in becoming a
useful alternative to polymeric hydrogels.2 For example, su-
pramolecular hydrogels are being explored to serve as scaffolds
for regenerative medicine,3 wound healing,4 biomineralization,5
vehicles for controlled drug release,6 matrices for protein micro-
arrays,7 a low-cost platform for screening enzyme inhibitors or
enzyme detection,8 and components for enzyme mimetics.9
Forming a hydrogel is the first step in developing supramole-
cular hydrogels as useful soft materials. There are several different
ways to generate supramolecular hydrogels. A commonly used
method involves dissolving hydrogelators into an aqueous solu-
tionand changing the temperature, pH, orionicstrength to initiate
molecular self-assembly in water, resulting in hydrogelation.1
This kind of approach, which works well for most hydrogelators,
has some inherent disadvantages for certain hydrogelators, for
example, a hydrogelator with exceedingly low solubility (or unusually
high hydrophobicity) or having the potential to form precipitates
instead of a hydrogel as a result of the change in temperature,
ionic strength, or pH. These molecules have the potential to self-
assemble in water despite their poor aqueous solubility. One
approach is to dissolve them in a polar organic solvent and then
mix this solution with water to form nanostructures or hydrogels.
For example, in work on the self-assembly or hydrogelation of
NH2-Phe-Phe-COOH,10 Fmoc-Phe-Phe,11 Fmoc-Phe(F5),12
GSH-pyrene,13 and NH3þ-Phe-Phe-CO-NH2,14 an organic sol-
vent (usually 1,1,1,3,3,3-hexafluoro-2-propanol or DMSO) is
always necessary to assist the dissolution of these compounds.
Despite its effectiveness, this approach inevitably brings small
amount of organic solvent into the hydrogels, which changes, if
not completely disturbs, the biocompatibility or rheological
behavior of the resulting hydrogels.
To explore new methods for making hydrogels, we and others
have been developing new ways to induce hydrogelation via
chemical or enzymatic conversion. For example, the phosphor-
ylation of tyrosine residues on hydrogelators offers precursors
that are soluble at physiological pH. Then a phosphatase can
hydrolyze the phosphoric monoester and convert the precursors
to less soluble but amphiphilic hydrogelators, which self-assemble
in water to form supramolecular nanofibers and result in hydro-
gels.15 Besides the route of dephosphorylation, other chemical or
enzymatic paths should also be suitable for the generation of
supramolecular hydrogels, which have been less explored.16
In this work, we explored the paths for generating the hydrogel
of a small molecule (2). Because of its high hydrophobicity, neither
the change in pH nor the change in temperature creates the hydro-
gel of 2. However, a simple chemical modification of 2 offered
† Part of the Supramolecular Chemistry at Interfaces special issue.
*Corresponding author. Tel: 1-781-736-5201. Fax: 1-781-736-2516. E-mail:
bxu@brandeis.edu.
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Rajagopal, K.; Branco, M.; Salick, D.; Rughani, R.; Pilarz, M.; Lamm, M. S.;
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1510 DOI: 10.1021/la103982e
Published on Web 12/08/2010
Langmuir 2011, 27(4), 1510–1512

Zhao et al.
Article
Figure 1. (A) Synthesis route to 2 and 3. (B) Reactions to convert
the precursor (3) to the hydrogelator (2) under basic conditions
(pH9) toaffordgelIorviahydrolysiscatalyzedbyesterase(1U/μL,
10 μL) at pH 7.5 to give gel II (optical images: left, normal; right,
through a pair of crossed polarizers).
Figure 2. TEM images of the matrices in (A) gel I and (B) gel II
(aged for 7 days).
molecule 3 (i.e., the precursor of 2) with excellent solubility at phys-
iological pH, and a chemical (i.e., a strong base) or a biological
catalyst (i.e., anesterase) triggeredthe hydrolysis of the carboxylic
ester bond of 3 to produce 2, which self-assembled into a supra-
molecular hydrogel. Because 2 lacks a carboxylic acid group or an
amine group, the hydrogel of 2 exhibits extraordinary stability
over a wide pH range, which is crucial for some applications of
biomaterials.17 In addition, unlike most of the supramolecular
hydrogels, the hydrogel of 2 is anisotropic (i.e., it exhibits bire-
fringence), which is associated with the order of the nanofibers.
Besides the identification of a novel hydrogelator, this work illus-
trates a powerfulstrategy for evaluating the ability of hydrophobic
molecules to form supramolecular hydrogels, which may provide
a new direction for designing supramolecular materials based on
small-molecule therapeutics because a large number of them are
quite hydrophobic.
Figure 1 shows the structures and the syntheses of 2 and 3. Two
cycles of activation of 1 by N-hydroxysuccinimide (NHS) and
coupling with the amine group on phenylalanine and ethanol-
amine, respectively, give 2 in 91% yield (Scheme S1). Finally, the
reaction between succinic anhydride and 2 results in 3, which
exhibits a drastically increased aqueous solubility (>20 mg/mL)
compared to that of 2 (<20 μg/mL). After obtaining pure com-
pounds 2 and 3, we tested the ability of 2 to form a hydrogel and
found that it failed to form a hydrogel via changing the tempera-
ture or adjusting the pH. Unlike 2, compound 3 dissolves easily in
a weak basic solution (pH 7.5) at the concentration of 0.8% (w/v).
The addition of a small amount of base (NaOH, 1.0 M, 10 μL)
Figure 3. (A) Optical images of the gels of 2 immersed in solution
with different pH values. (B) Dynamic strain sweeps (at 6.28 rad/s)
of aged gel I (400 μL, 0.8% (w/v)), without treatment and immer-
sion in 1.0 M HCl (20 mL), 1.0 M NaOH (20 mL), and 1.0 M NaCl
(20 mL) for half an hour. (C) Dynamic frequency sweeps of
corresponding gel I.19
to reach pH 9 or esterase (1 U/μL, 10 μL) in 400 μL of the solution
of 3 causes the hydrolysis of 3. The loss of succinic acid from
3 affords 2 in almost quantitative yield (Figure S1), which self-
assembles in water to result in a clear hydrogel that shows strong
birefringence (Figure 1B) without a noticeable pH change. The
use of NaOH to hydrolyze 3 helps to determine the hydrolysis rate
(Figure S2) and to estimate the critical gelation concentration
(CGC) of 2 to be 0.16% (w/v) (Figure S3). We designate the
hydrogels formed by the addition of NaOH and esterase as gels
I and II, respectively.
We used a dynamic time sweep experiment to determine
the gelling point upon the addition of base or enzyme. The gelling
points of gels I and II were about 1 and 38 h, respectively
(Figure S4), which match the observation in the TEM images of
(17) (a) Hong, Y. H.; McClements, D. J. J. Agric. Food Chem. 2007, 55, 5653–
5660. (b) Frenkel-Mullerad, H.; Avnir, D. J. Am. Chem. Soc. 2005, 127, 8077–8081.
(c) Wang, J.; Musameh, M.; Mo, J. W. Anal. Chem. 2006, 78, 7044–7047.
Langmuir 2011, 27(4), 1510–1512
DOI: 10.1021/la103982e 1511

Article
Zhao et al.
the two hydrogels (Figure 2). According to the TEM results, gel I,
which formed faster, consists of short fibers (<500 nm) with more
randomly interwoven networks (Figure 2A). Gel II, which
formed more slowly, containing long fibers (>2 to 3 μm) with
less-dense networks (Figure 2B).
In addition, increasing the quantity of reagents for ester bond
hydrolysis can speed up the hydrolysis and thus gelation. Upon
the addition of a larger amount of NaOH, gel I forms within 3 min
(video in Supporting Information). Besides controlling the gela-
tion speed, different hydrolysis routes may be applied to different
applications. For example, gelation catalyzed by esterase can pro-
ceed under physiological condition, which will be useful in encap-
sulating biomacromolecules. Moreover, we can tailor the proper-
ties of the hydrogels with different microstructures for different
applications by adding different catalysts at various quantities.
When immersed in both highly acidic and highly basic solu-
tions, the hydrogels of 2 showed excellent stability (Figure 3A).
To understand the stability of the hydrogel of 2 further, we quan-
tified its rheological properties after thegels were treatedwith HCl
(1.0 M) or NaOH (1.0 M). The dynamic strain sweeps and dy-
namic frequency sweeps reveal that aged gel I retains its rheolo-
gical properties after immersion in solutions (20 mL) with various
pH and a salt solution (20 mL) for half an hour (Figure 3B,C).
The gel in basic solution (pH 14) maintains the G0 and G00 values
that are comparable to those of native gel I, although the critical
strain seemed to decrease from 40 to 4%. The acid-treated gel
shows anincrease inthe storage modulus of up to at least 2.5 times
the G0 value of native gel I. Besides, upon treatment of the 1.0 M
HCl solution, the volume of gel I changes little (Figure S5).
However, gel I treated with a 1 M NaCl aqueous solution exhibits
a slight decrease in viscoelasticity. The above results suggest that
the acid may increase the hydrogen bonding among the hydro-
gelators and water.
Because the formation of gel II is slow enough to allow us to
monitor the optical and spectroscopic changes over time, we syn-
chronized the time-dependent CD spectra and the time-dependent
birefringence of gel II to examine the gel-formation process
(Figure 4). In the first 12 h, the CD data hardly showed peaks
below 210 nm and we observed no birefringence. However, after
that, the birefringence as evidenced by the negative peak at
around 209.6 nm and the positive peak at 195.8 nm also appeared.
Afterward, the birefringence increases and these two peaks
become larger and sharper. According to Boden and co-workers’
pioneering work,18 the appearance of these two peaks correspond
to the formation of the β-sheet. In addition, the increase of
anisotropy likely contributes to the higher positive peak at 230 nm.
Therefore, these results suggest that the birefringence is associated
with the formation of the β-sheet (Figures 4 and S6).
Figure 4. (A) Optical images of gel II (formed by the catalysis
of esterase) under cross-polarized light at different time points and
(B) the corresponding CD spectra.
In conclusion, we have demonstrated that the controlled hydro-
lysis of a carboxylic ester bond by a base or an enzyme can act as a
simple trigger to initiate self-assembly and form supramolecular
hydrogels, even if the molecule itself is unable to form hydrogels
by directly changing the pH or temperature. The replacement of a
carboxylate group with a hydroxyl group on the hydrogelator not
only results ina supramolecular hydrogel that is stable over a wide
pH range but also offers a soft supramolecular material that is
insensitive to ionic strength. These properties may be particularly
useful in designing a robust drug release system that can maintain
a constant release rate against abrupt changes in the environment,
which is a subject that is currently under investigation.
Acknowledgment. We are grateful for financial support from
the NIH (R01CA142746-01) a start-up grant from Brandeis
University, and an HFSP grant (RGP0056/2008). We thank the
EM facility at Brandeis University for assistance.
Supporting Information Available: The experiment details,
NMR, hydrolysis product analysis, hydrolysis rate, CGC
test, dynamic time sweep, video of fast gelation, swelling test,
and the normalized intensities of CD. This material is available
free of charge via the Internet at http://pubs.acs.org.
(18) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; McLeish, T. C. B.; Nyrkova, I.;
Radford, S. E.; Semenov, A. J. Mater. Chem. 1997, 7, 1135–1145.
(19) For the dynamic frequency sweeps of gel I, the strain on the gel without any
treatment is 0.15% and the strains on gel I after acid treatment, base treatment, and
salt treatment were 0.125, 0.158, and 0.198%, respectively
1512 DOI: 10.1021/la103982e
Langmuir 2011, 27(4), 1510–1512