Aromatic-aromatic interactions enhance interfiber contacts for enzymatic formation of a spontaneously aligned supramolecular hydrogel

Article abstract of DOI:10.1021/ja4127399

Anisotropy or alignment is a critical feature of functional soft materials in living organisms, but it remains a challenge for spontaneously generating anisotropic gel materials. Here we report a molecular design that increases intermolecular aromatic-aromatic interactions of hydrogelators during enzymatic hydrogelation for spontaneously forming an anisotropic hydrogel. This process, relying on both aromatic-aromatic interactions and enzyme catalysis, results in spontaneously aligned supramolecular nanofibers as the matrices of a monodomain hydrogel that exhibits significant birefringence. This work, as the first example of monodomain hydrogels formed via an enzymatic reaction, illustrates a new biomimetic approach for generating aligned anisotropic soft materials.

Full text of DOI:10.1021/ja4127399

Communication
pubs.acs.org/JACS
Aromatic−Aromatic Interactions Enhance Interfiber Contacts for
Enzymatic Formation of a Spontaneously Aligned Supramolecular
Hydrogel
Jie Zhou, Xuewen Du, Yuan Gao,† Junfeng Shi, and Bing Xu*
Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States
S
* Supporting Information
ing because the self-assembly of hydrogelators usually affords
nanofibers,9a−c which is inherently anisotropic. However, these
ABSTRACT: Anisotropy or alignment is a critical feature
of functional soft materials in living organisms, but it
remains a challenge for spontaneously generating aniso-
tropic gel materials. Here we report a molecular design
that increases intermolecular aromatic−aromatic interac-
tions of hydrogelators during enzymatic hydrogelation for
spontaneously forming an anisotropic hydrogel. This
process, relying on both aromatic−aromatic interactions
and enzyme catalysis, results in spontaneously aligned
supramolecular nanofibers as the matrices of a mono-
domain hydrogel that exhibits significant birefringence.
This work, as the first example of monodomain hydrogels
formed via an enzymatic reaction, illustrates a new
biomimetic approach for generating aligned anisotropic
soft materials.
nanofibers, being long and flexible, not only physically cross-
link to form the matrices of gels but also adopt random
entanglement, which results in isotropic hydrogels in most
cases.8a Encouragingly, several exceptional examples of supra-
molecular hydrogels exhibit birefringence, thus offering useful
hints for creating anisotropic hydrogels. For example, Stupp
and co-workers demonstrated that a thermal pathway can
convert isotropic solutions of peptide amphiphiles to a strongly
birefringent hydrogel, in which molecules self-assemble to form
large arrays of aligned nanofibers.4a Moreover, the addition of
calcium ions into the solution of a phosphate containing
peptide amphiphile results in a gel that exhibits birefringence,
which coincides with the parallel alignment of the nanofibers
shown in the transmission electron micrograph (TEM) of the
gels.10 These results and other examples11a,b suggest that the
enhancement of interfiber interactions should be a reliable
molecular approach to induce alignment of nanofibers and thus
generate anisotropy.
nisotropy or alignment is a critical feature of functional
A
materials in living organisms or man-made systems. For
example, muscle cells rely on sophisticated anisotropic protein
filaments to function, and liquid crystal display is based on the
electrical static alignment of a simple anisotropic molecule (e.g.,
4-cyano-4′-pentylbiphenyl1). Recognizing the importance of
anisotropy, materials scientists are striving to create high quality
anisotropic materials. While generating anisotropic materials
from inorganic solids is relatively easy and successful,2a,b the
introduction of anisotropy to synthetic soft organic materi-
als,3a−d particularly hydrogels,4a−c remains less developed and a
considerable challenge despite the fact that biological
anisotropic soft materials with substantial complexity prevail
in living organisms.5 Although several approaches have shown
effectiveness for inducing anisotropy in gels, including the use
of an electrical field,6 the inclusion of liquid crystals,3c,d and the
utilization of mechanical stretching7a−c or shear force from
flow,4a each process still has its limitation for creating
anisotropy in hydrogels because hydrogels contain a large
amount of water that usually conducts electricity, excludes
common liquid crystals, and favors relaxation to the isotropic
state. Thus, better approaches are needed for spontaneously
generating inherently anisotropic hydrogels.
Although ionic forces10,11b or overlaps between alkyl
chains11a are able to increase the interfiber interactions, ionic
interactions are intrinsically isotropic and interactions between
alkyl chains are relatively weak and inefficient. Therefore, we
decided to explore the use of aromatic−aromatic interactions to
enhance interfiber interactions that are necessary for creating
anisotropic supramolecular hydrogels because (i) aromatic−
aromatic interaction is not only stronger than the van der
Waal’s interaction (London dispersion force) between alkyl
chains but also inherently directional due to the plane-to-plane
or edge-to-plane orientation;12 (ii) nature has extensively used
aromatic−aromatic interaction as a stabilizing force for
generating ordered structures in proteins;12 and (iii) aromatic
rings have relatively compact volumes; thus, the interactions
between aromatic rings lead to a more predictable and efficient
self-assembly of the molecules in aqueous phase for the
formation of mechanically strong or stable supramolecular
hydrogels.13
Besides the use of proper interfiber interactions, it is
necessary to choose an appropriate pathway for molecular
self-assembly. Instead of using a thermal pathway,4a we decided
to use an enzymatic pathway because cells use enzymatic
conversion to form anisotropic hydrogels5 and the enzymatic
Based on the anisotropic hydrogel in nature (e.g., corneal
stroma formed mainly by regularly arranged collagen fibers5),
one attractive approach is to use molecular self-assembly to
generate anisotropic structures that immobilize water and form
hydrogels. In fact, recent advances in the development of
supramolecular gels8a−d make this approach particular promis-
Received: December 16, 2013
Published: February 10, 2014
© 2014 American Chemical Society
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Communication
self-assembly process has emerged as a powerful method for
generating ordered nanostructures14 and supramolecular hydro-
gels for a wide range of applications.15a,b For example,
enzymatic self-assembly has led to supramolecular hydrogels
outside cells16 and inside cells,17a,b in vitro and in vivo,18 and
resulted in highly stable hydrogels that are unavailable via other
pathways (e.g., thermal or pH).2a,4c,19 Recently, enzyme
regulated self-assembly has provided a route for controlled
drug release20a,b and for the exploration of dynamic self-
assembly.21 Despite these advancements, it remains unknown
whether the enzymatic pathway can lead to aligned
monodomain supramolecular hydrogels.
To address the above-mentioned question, we designed two
pairs of precursor/hydrogelator (i.e., 1a/1b and 2a/2b)
differing only in one phenylalanine (Phe) residue and examined
their enzymatic hydrogelation by using polarized optical
microscopy, transmission electron microscopy (TEM), and
rheology. Our study shows that the addition of an enzyme (e.g.,
alkaline phosphatase (ALP)) into the solution of 1a affords an
aligned monodomain supramolecular hydrogel of 1b, which
consists of aligned nanofibers (Figure 1). Having one less
precursor 2a, as an analog of 1a, consists of Nap, Phe-Phe, Lys,
and pTyr. Nap enhances intermolecular aromatic−aromatic
interactions;22 the phenylalanine residues (Phe), besides
providing aromatic−aromatic interactions, constitute the
peptide backbone with Lys to provide the donors and acceptors
for intermolecular hydrogen bonds. Since ALP catalytically
dephosphorylates Tyr to Tyr to form the hydrogelators, 1b
p
and 2b, the phosphate group on Tyr acts as a trigger for
enzymatic hydrogelation.15a The hydrogelator 1b, bearing three
phenylalanine residues, likely has stronger intermolecular
aromatic−aromatic interactions than 2b (bearing two phenyl-
alanine residues) does. After the molecular design, we used
solid phase peptide synthesis (SPPS)23 to synthesize 1a, 2a, 1b,
and 2b (Scheme S1).
As a soluble precursor, 1a (or 2a) (4 mg) dissolves in
distilled water (0.5 mL) to a final concentration of 0.8 wt % and
pH = 7.4. After the addition of ALP (2U/mL), the solution of
1a (or 2a) transforms to a transparent hydrogel (Figure S1,
here denoted as Gel1b (or Gel2b)) within 2 h because the
precursor 1a (or 2a) becomes the corresponding hydrogelator,
1b (or 2b). Gel1b, being placed between the cross polarizers,
exhibits little birefringence when the vial is at 90° and 0° with
polarizers (Figure 2A) but displays significant birefringence
Figure 2. Optical images of Gel1b and Gel2b, formed by treating the
solutions of 1a and 2a with ALP (2 U/mL) at the concentration of 0.8
wt % and pH of 7.4 overnight. The images are taken with the vials or
capillaries (d = 0.3 mm) placed between cross polarizers, illuminating
by ambient light. (A, B) Gel1b in a vial and (C, D) Gel2b in a vial. (E,
F, G) Gel1b formed in two capillaries that are placed at three different
angles with the cross polarizers.
Figure 1. (A) Illustration of aromatic−aromatic interactions, in a
hydrogel (Gel1b) formed by treating the solution of a precursor (1a)
with an enzyme (ALP), to enhance interfiber interactions that favors
alignment of nanofibers and results in an inherently anisotropic
hydrogel that causes optical retardance. (B) Structures of two
hydrogelators, differing only in the number of phenylalanine residues.
when the vial is at 45° with the polarizers (Figure 2B). This
result indicates that the nanofibers of 1b in Gel1b have largely
uniform alignment to result in a significant birefringence. In
contrast, Gel2b hardly shows any birefringence in either of the
orientations (90° and 0° in Figure 2C and 45° in Figure 2D).
This result indicates that the nanofibers of 2b in Gel2b likely
have random orientations. To further verify the alignment of
nanofibers in Gel1b, we carried out the enzymatic gelation of
Gel1b in two capillaries with a diameter of 0.3 mm. As shown in
Figure 2E, F, Gel1b barely exhibits birefringence when the
capillaries are at 90° and 0° with the polarizers (Figure 2E) but
displays bright birefringence when the capillaries are at 45° with
the polarizers (Figure 2F). In addition, light extinguishes at the
crosspoint of two capillaries containing Gel1b (Figure 2G),
confirming uniform alignments of the nanofibers of 1b in Gel1b.
Although the change of the solution pH of 1b (or 2b) also
results in a hydrogel (Figure S2A (or S3A), denoted as Gel1b′
(or Gel2b′)) at the concentration of 0.8 wt % and pH of 7.4,
Gel1b′ shows sporadic, random, and orientation-independent
birefringence (Figure S2B and S2C), and Gel2b′ hardly shows
phenylalanine residue, 2b forms a nonbirefringent hydrogel that
consists of a network of randomly oriented nanofibers. In
addition, the hydrogel of 1b (or 2b) formed by the change of
pH exhibits little birefringence and consists of few aligned
nanofibers. Thus, this work, as the first example of
monodomain supramolecular hydrogels formed by an enzy-
matic reaction, illustrates a new, effective approach for
generating anisotropic supramolecular soft materials, which
ultimately may lead to the formation of aligned nanostructures
directly in a cellular environment.
Figure 1B shows the detailed molecular design of the two
pairs of precursor/hydrogelator. The precursor 1a, as a
pentapeptidic derivative, consists of a (naphthalene-2-ly)acetyl
group (Nap), three phenylalanine residues (Phe-Phe-Phe), a
lysine (Lys), and a tyrosine phosphate moiety (pTyr). The
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Communication
any birefringence (Figure S3B and S3C). These results indicate
enzymatic hydrogelation as a feasible and effective pathway to
generate an aligned, monodomain supramolecular hydrogel.
To quantify the birefringence in the hydrogels, we used
polarized microscopy to image the hydrogels made of different
samples (i.e., 1b and 2b) and by different pathways (i.e.,
enzyme or pH). Birefringence is a material property that
derives from molecular alignment, which results in a fast and a
slow axis in the material. The polarization of light changes due
to a differential phase shift between two orthogonal polarized
components, one of which, being parallel to the fast axis, travels
faster than the other that is parallel to the slow axis. Retardance
(Δn) signifies the differential phase shift between two
wavefronts of the orthogonal components that have traveled
through a birefringent material and is typically measured as a
distance in the unit of nm (Figure 1A). Polarized microscopy
with the OpenPolScope method24 measures the retardance of
every resolved point of specimen and converts the results of
computation into a color gradient of the retardance images.
Thus, we used PolScope to obtain retardance images of Gel1b,
Gel2b, Gel1b′, Gel2b′, and a solution of 1a or 2a. As shown in
Figure 3A, the thin film of Gel1b contains largely uniform
Figure 4. TEM images of the nanofiber matrices of (A) Gel1b and (B)
Gel2b. The scale bar is 250 nm.
entangled nanofibers (d = 8 2 nm) (Figure S6A and S6B).
Gel2b′, however, contains entangled nanofibers (d = 8 2 nm)
to form a network, exhibiting the same morphology as that of
Gel2b (Figure S6C and S6D). The agreement between the
TEM images and PolScope images confirms that the alignment
of the nanofibers formed during enzymatic hydrogelation is
responsible for birefringence of Gel1b.
To further verify the results in TEM images, we also
compared the rheological properties of the hydrogels. Figure 5
Figure 5. Rheological characterization of Gel1b, Gel2b, Gel1b′, and
Gel2b′. (A) The strain dependence of the dynamic storage (G′) and
loss storage (G″) is taken at a frequency equal to 6.28 rad/s, and (B)
the frequency dependence is taken at a strain equal to 0.99%.
Figure 3. Polarized optical microscopy retardance images (scale bar =
100 μm) of (A) Gel1b, (B) Gel2b, (C) Gel1b′, (D) Gel2b′. The images
are taken with a sample thickness of 235 μm and concentration of 0.8
wt %.
shows the strain and frequency dependence of dynamic storage
moduli (G′) and loss moduli (G′′) of Gel1b, Gel2b, Gel1b′, and
Gel2b′ at a concentration of 0.8 wt %. The values of the storage
moduli (G′) of all four gels are larger than those of their loss
moduli (G′′), indicating that all the samples behave as
viscoelastic gel materials. The values of G′ of the hydrogels
change little during the frequency sweep (from 0.1 to 200 rad/
s) (Figure S7), suggesting that the matrices of those gels have
good tolerance to external shear force. In agreement with TEM
data, Gel2b, which consists of entangled nanofibers (more cross-
linking), shows stronger mechanical strength than Gel1b, which
contains parallel aligned nanofibers (less cross-linking). On the
other hand, when both of them have similar nanofiber networks
(Figure S6), Gel1b′ exhibits a slightly higher storage moduli
than Gel2b′, agreeing with that hydrogelator 1b has one more
phenylalanine residue to provide stronger aromatic−aromatic
interactions than 2b does.
In conclusion, this communication describes a rational design
that utilizes aromatic−aromatic interactions to promote
interfiber contacts between nanofibers and to drive the
alignment of nanofibers for producing inherently anisotropic
supramolecular hydrogels via enzyme catalyzed molecular self-
assembly. Although direct evidence of aromatic−aromatic
interfiber interaction remains to be established, the crystal
structure of the self-assembly motif (Nap-Phe-Phe)22 in 1
strongly supports the notion of aromatic−aromatic interfiber
domains (with the sizes of hundreds of micrometers) that
exhibit a retardance of approximately 35 nm or larger. Gel1b′,
however, exhibits mostly multiple domains that have retardance
between 0 and 25 nm (Figure 3C). As shown in Figure 3B and
3D, Gel2b and Gel2b′, under PolScope, give a retardance value
of almost 0 nm, confirming that Gel2b and Gel2b′ are
nonbirefringent. As expected, a solution of 1a or 2a at the
concentration of 0.8 wt % and pH of 7.4 are nonbirefringent, as
proven again by PolScope imaging (Figure S4). These results
further support that the combination of strong aromatic−
aromatic interactions and enzymatic hydrogelation facilitates
the formation of anisotropic supramolecular hydrogels.
Transmission electron microscopy (TEM) also confirms the
orientation orders of the nanofibers in Gel1b. As shown in
Figure 4A, the nanofibers in Gel1b have diameters (d) of 8
2
nm and form exceptionally long, aligned bundles that stretch
over many micrometers (Figure S5), suggesting strong
interfiber interactions. In contrast, TEM shows that Gel2b
contains a large amount of randomly entangled nanofibers (d
= 8 2 nm) that form a typical network (Figure 4B) as those
found in conventional supramolecular hydrogels. A TEM image
of Gel1b′ indicates a mixture of parallel aligned nanofibers and
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interactions. In addition, intermolecular aromatic−aromatic
interaction from the overlapping of phenyl and/or naphthyl
groups, which are responsible for forming single molecular
width nanofibers,25 should favor interfiber interactions. The
alignment of the nanofibers likely stems from the stereo-
chemical cooperation between phosphatases and the precur-
sors/hydrogelators because the use of the enantiomer of 1a for
enzymatic hydrogelation hardly results in the alignment of
nanofibers (Figure S7). Since the hydrogel directly prepared
with 1b by a change in pH exhibits little birefringence and
consists of few aligned nanofibers, enzymatic conversion is
indispensable for the alignment of the nanofibers. The increase
of the concentration of enzyme speeds up the gelation process,
but shows little influence on birefringence or alignment. Our
work, for the first time, illustrates PolScope imaging is a useful
and effective method to study anisotropy of supramolecular
hydrogels. By establishing enzyme catalysis as a new pathway to
complement other processes for generating inherently aniso-
tropic hydrogels, this work may lead to the production of
aligned nanostructures in biological systems or living
organisms.
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ASSOCIATED CONTENT
* Supporting Information
The details of the synthesis, NMR spectra and LC-MS data for
all compounds, and rheological data. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
bxu@brandeis.edu
■
(18) Yang, Z.; Liang, G.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2006,
128, 3038.
Present Address
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Chem. Soc. 2009, 131, 11286.
(20) (a) Gao, Y.; Kuang, Y.; Guo, Z.-F.; Guo, Z.; Krauss, I. J.; Xu, B.
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L.; Eelkema, R.; Koper, G. J. M.; van Esch, J. H. J. Am. Chem. Soc.
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†Eunice Kennedy Shriver National Institute of Child Health
and Human Development, National Institutes of Health, 13
South Drive, Bethesda, Maryland 20892, United States.
Notes
The authors declare no competing financial interest.
(21) Debnath, S.; Roy, S.; Ulijn Rein, V. J. Am. Chem. Soc. 2013, 135,
16789.
ACKNOWLEDGMENTS
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This work was partially supported by NIH (R01CA142746)
HFSP and a start-up fund from Brandeis University. We thank
Brandeis EM and Optical Imaging facilities for TEM and Prof.
Z. Dogic, Dr. Rudolf Oldenbourg, and Mr. Mark Zakhary for
technical assistance on polarized microscopy.
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