Molecular nanofibers of olsalazine form supramolecular hydrogels for reductive release of an anti-inflammatory agent

Article abstract of DOI:10.1021/ja109269v

Conjugation of tripeptide derivatives with olsalazine, a clinically used anti-inflammatory prodrug, yields small molecules that self-assemble in water to form supramolecular hydrogels that undergo a gel-to-sol phase transition upon reduction, resulting in the controlled release of 5-aminosalicylic acid as the anti-inflammatory agent. This methodology will ultimately lead to new biomaterials for site-specific drug delivery.

Full text of DOI:10.1021/ja109269v

Published on Web 12/01/2010
Molecular Nanofibers of Olsalazine Form Supramolecular Hydrogels for
Reductive Release of an Anti-inflammatory Agent
Xinming Li, Jiayang Li, Yuan Gao, Yi Kuang, Junfeng Shi, and Bing Xu*
Department of Chemistry, Brandeis UniVersity, 415 South Street, Waltham, Massachusetts 02454, United States
Received October 14, 2010; E-mail: bxu@brandeis.edu
ways of creating supramolecular hydrogels as smart materials for
controlled drug release at specific sites or organs in a biological
system.
Because colonic microflora secrete azo reductase to reduce the
azo group into the corresponding amine, use of olsalazine (a
Abstract: Conjugation of tripeptide derivatives with olsalazine,
a clinically used anti-inflammatory prodrug, yields small molecules
that self-assemble in water to form supramolecular hydrogels that
undergo a gel-to-sol phase transition upon reduction, resulting
in the controlled release of 5-aminosalicylic acid as the anti-
inflammatory agent. This methodology will ultimately lead to new
biomaterials for site-specific drug delivery.
substrate of azo reductase) as a prodrug achieves colon-specific
drug delivery17 via catalytic generation of mesalazine [or 5-ami-
nosalicylic acid (5-ASA)] inside the colon at the site of inflam-
mation.18 This opportunity for reductive degradation of azo
compounds by colonic microflora has led to the development of a
score of polymeric azo compounds that have found application for
The paper describes a supramolecular hydrogel as a potential
colon targeting, as the reduction and subsequent splitting of the
biomaterial for site-specific drug release. Biomaterials derived from
azo bond occurs only in the large intestine.19 Encouraged by these
synthetic or biological polymeric hydrogels have found widespread
results, we developed an olsalazine-containing supramolecular
applications in biomedical engineering, ranging from tissue repair
hydrogel as a candidate for a smart biomaterial for controlled
to regenerative medicine and drug delivery.1 These polymer-based
release. Specifically, we synthesized hydrogelator 1 by using a
hydrogels, however, still have several inherent shortcomings, such
as relatively slow degradation, unintended immune responses, and
tripeptide derivative that consists of a naphthyl group, two
phenylalanines, and one modified lysine residue carrying an
the generation of undesirable byproducts.2 On the other hand,
olsalazine moiety in the side chain. 1 self-assembles to form a
supramolecular hydrogels3 formed by low-molecular-weight gela-
hydrogel under mildly acidic conditions. The reduction of olsalazine
tors4 that self-assemble in water through noncovalent interactions
not only leads to a gel-to-sol phase transition but also releases
have attracted considerable attention because they exhibit several
5-ASA. Through direct incorporation of the prodrug into the
unique merits, such as synthetic economy, biocompatibility, low
nanofibers, this supramolecular hydrogel demonstrated a new way
toxicity, inherent biodegradability, and most importantly, fast
to encapsulate the prodrug and release the active ingredients.
thermally reversible formation-dissociation processes.5 These
Because a large pool of prodrugs exists, this work contributes to
advantages make supramolecular hydrogels a promising alternative
the future design of new smart biomaterials based on supramolecular
for polymeric hydrogels. Among the molecules that can act as the
chemistry20 and prodrugs.
building blocks for supramolecular hydrogels, peptide-based hy-
drogelators6 are the usual candidates because of their biological
relevance, the well-established synthetic chemistry (i.e., solid-phase
synthesis),7 and the capability to produce a large set of diverse
functional molecules from a small array of residues. There are many
examples of peptide-based functionalized building blocks for
making nanofibers and generating hydrogels. The nanofibers of
peptide amphiphile molecules can display a high density of epitopes
for regulating the differentiation of neuron progenitor cells8 or
guiding cartilage regeneration.9 A supramolecular hydrogel self-
assembled from lysine-containing short peptides has been shown
to exhibit inherent antibacterial activity.10 Self-complementary
oligopeptides form hydrogels for cell culture and cytokine release.11
Amino acid-functionalized hydrogel particles release protein when
triggered enzymatically.12 A small peptide conjugated with ꢀ-lactam
Figure 1. Illustration of drug release from the olsalazine-containing
supramolecular hydrogel upon reduction.
is transformed into a hydrogelator by the catalysis of a ꢀ-lacta-
mase.13 A low-molecular-weight gelator containing amino acid
Figure 1 illustrates the structure of hydrogelator 1, which contains
a short peptide motif and an olsalazine moiety. We synthesized
moieties confers liquid-crystalline (LC) gels.14 Photosensitive
spiropyran linking with dipeptide leads to a supramolecular hydrogel
that responds to both light and ligand-receptor interaction.15
Despite these advances, the application of supramolecular hydrog-
elators in controlled drug release has been less explored,16 and there
has been even less exploration of supramolecular hydrogels for site-
specific drug release. It is necessary and important to explore new
small-molecule hydrogelator 5, which is a tripeptide derivative made
by conjugating 2-(naphthalen-2-yl)acetic acid with Phe-Phe-Lys.
In our recent study,21 we found that tripeptide derivative 5 forms
a hydrogel at a very low critical gelation concentration (0.8 wt %).
Conjugation of 5 to an olsalazine moiety through the ε-amino group
of the lysine residue afforded 1, which we expected to form a stable
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C O M M U N I C A T I O N S
supramolecular hydrogel as the prodrug reservoir that upon azo
reduction would disassemble and release 5-ASA.
or D-1 was transformed into a light-yellow suspension (Figure 2).
HPLC and LC-MS analysis of the suspension (Figures S1 and
S2) confirmed the conversion of 1 into the corresponding products
2 (tR ) 18.2 min) and 5-ASA (tR ) 4.1 min). The identification of
5-ASA validates that this supramolecular hydrogel can act as a
reservoir of the prodrug and release the 5-ASA upon reduction of
the azo bond.
Scheme 1. Synthetic Scheme for Olsalazine-Containing
Hydrogelator 1
Transmission electron microscopy (TEM) helped to evaluate the
extent of the self-assembly of hydrogelator 1 during different stages
of the gel-to-sol transition. As shown in Figure 2, the hydrogelators
L-1 and D-1 self-assemble to afford nanofibers with widths of 11
and 13 nm, respectively, and lengths of more than several
micrometers (Figure 2C,G). In addition, the hydrogelator D-1 shows
nanofibers with a right-handed helical structure (Figure S6). These
nanofibers constitute the matrices (in the form of bundles or
networks) of the hydrogels of 1. The TEM images of the negative
staining suspensions in Figure 2B,F indicate the loss of the long
nanofibers after reductive cleavage of the azo bond, in agreement
with the observation that 2 failed to act as a hydrogelator. The
dissociation of the three-dimensional networks of the nanofibers
upon reduction indicates that the hydrogels of 1 should be able to
release 5-ASA upon the action of azo reducatase.17
Circular dichroism (CD) studies provided further molecular
insight into the self-assembly of 1 and the gel-to-sol transition upon
reduction. The hydrogelator L-1 in the gel phase gives a CD
spectrum with a ꢀ-sheet signature, as evident by negative bands at
205 nm and positive bands at 195 nm (Figure S3).22 Upon
reduction, the gel turns into the sol as a result of the conversion of
hydrogelator L-1 into compound L-2 accompanied by release of
5-ASA. The CD signal of the ꢀ-sheet decreased significantly,
indicating that L-2 self-assembles less efficiently than hydrogelator
L-1 because of the loss of 5-ASA. The reduction of D-1 generates
D-2 and also exhibits a decrease in the signal between 190 and 204
nm that is similar to the decrease of the ꢀ-sheet signal for L-1
(Figure S3).22 The hydrogel of D-1 exhibits a strong CD band
around 480 nm that is far from the chromophoric absorption region
(∼360 nm) of olsalazine. This peak likely originates from a
mesophase of D-1,23 which agrees with the birefringence of the
hydrogel of D-1 (Figure S6).
Scheme 1 shows the synthetic route to 1. The HBTU-activated
compound 3 reacts with 5 to afford hydrogelator 1 in 48% yield
after purification by flash column chromatography. After obtaining
1, we tested its ability to form a hydrogel in water by adjusting the
pH. Typically, 6.0 mg of 1 dissolved in 0.50 mL of water to give
a clear solution, after which a change in pH to 5.0 resulted in a
viscous suspension. Ultrasound sonication of the suspension for 2
min or an increase of its temperature to ∼60 °C followed by cooling
to ambient temperature afforded a transparent, yellow gel (Figure
2). This experiment demonstrated that 1 is an effective hydrogelator
that forms a stable gel in water at a concentration of 1.2 wt %. In
order to further confirm that the naphthyl group is necessary for
compound 1 to form the hydrogel, we replaced the naphthyl group
with an acetyl group. We found that the molecule acetyl-FFK-
olsalazine (7) failed to form a hydrogel (Scheme S1 and Figure S5
in the Supporting Information). While the hydrogelator L-1 consists
of L-phenylalanine and L-lysine, the hydrogelator D-1 is made of
D-phenylalanine and D-lysine.
Figure 3. (A) Frequency dependence of the dynamic storage moduli (G′)
and loss moduli (G′′) of the hydrogel of L-1 and suspension of L-2. (B)
Frequency dependence of the dynamic storage moduli (G′) and loss moduli
(G′′) of the hydrogel of D-1 and suspension of D-2.
Figure 2. Optical images of (A, E) the hydrogels of (A) L-1 and (E) D-1
(1.2 wt %, pH 5.0) and (B, F) the suspensions of (B) L-1 and (F) D-1 after
the reduction reaction of the hydrogels. TEM images of (C, G) the matrices
of the hydrogels of (C) L-1 and (G) D-1 and (D, H) the broken fibers in the
suspensions of (D) L-2 and (H) D-2.
We used oscillatory rheology to examine the viscoelastic
properties of the hydrogels before and after reduction. Before the
reductive cleavage of the azo bond, the hydrogels of L-1 and D-1
both exhibit elastic properties of a solidlike material, as demon-
strated by the fact that the storage modulus (G′) was almost an
order of magnitude higher than the loss modulus (G′′) together with
the weak frequency dependence of the elasticity (Figure 3). After
the addition of the reductant, the value of G′ for the sample
decreased nearly 3 orders of magnitude, indicating that the material
In order to study reductant-mediated drug release from the
hydrogel, we dissolved 11 mg of sodium hydrosulfite in 0.2 mL of
pH 5 buffer and injected the reductant over the hydrogel. The final
concentration of hydrogelator 1 during the reduction reaction was
0.86 wt %. After incubation at 37 °C for 1 h, the hydrogel of L-1
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In conclusion, we have demonstrated that tripeptide derivatives
conjugated with olsalazine exhibit excellent self-assembly properties
and generate prodrug-containing supramolecular hydrogels. We
have also shown that reduction of the azo group can disrupt
the supramolecular hydrogels and release the active ingredient. The
use of D-peptides also should help preserve the stability of the
hydrogels against proteases in the upper gastronomical tract. Since
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Acknowledgment. This work was partially supported by the
Human Frontier Program (HFSP, RGP 0056/2008), a startup grant
from Brandeis University, and the NIH (R01CA142746-01) and
was assisted by the Brandeis University EM Facility.
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Supporting Information Available: Synthesis of hydrogelator 1,
HPLC traces and LC-MS data for the reduction of hydrogelator 1,
CD spectra, and rheological data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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