The conjugation of nonsteroidal anti-inflammatory drugs (NSAID) to small peptides for generating multifunctional supramolecular nanofibers/hydrogels

Article abstract of DOI:10.3762/bjoc.9.104

Here we report supramolecular hydrogelators made of nonsteroidal anti-inflammatory drugs (NSAID) and small peptides. The covalent linkage of Phe-Phe and NSAIDs results in conjugates that self-assemble in water to form molecular nanofibers as the matrices of hydrogels. When the NSAID is naproxen (1), the resultant hydrogelator 1a forms a hydrogel at a critical concentration (cgc) of 0.2 wt % at pH 7.0. Hydrogelator 1a, also acting as a general motif, enables enzymatic hydrogelation in which the precursor turns into a hydrogelator upon hydrolysis catalyzed by a phosphatase at physiological conditions. The conjugates of Phe-Phe with other NSAIDs, such as (R)-flurbiprofen (2), racemic flurbiprofen (3), and racemic ibuprofen (4), are able to form molecular hydrogels, except in the case of aspirin (5). After the conjugation with the small peptides, NSAIDs exhibit improved selectivity to their targets. In addition, the peptides made of D-amino acids help preserve the activities of NSAIDs. Besides demonstrating that common NSAIDs are excellent candidates to promote aromatic-aromatic interaction in water to form hydrogels, this work contributes to the development of functional molecules that have dual or multiple roles and ultimately may lead to new molecular hydrogels of therapeutic agents for topical use.

Full text of DOI:10.3762/bjoc.9.104

The conjugation of nonsteroidal anti-inflammatory
drugs (NSAID) to small peptides for
generating multifunctional
supramolecular nanofibers/hydrogels
Jiayang Li, Yi Kuang, Junfeng Shi, Yuan Gao, Jie Zhou and Bing Xu*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 908–917.
Department of Chemistry, Brandeis University, 415 South Street,
Waltham, MA 02454, USA
doi:10.3762/bjoc.9.104
Received: 26 February 2013
Accepted: 23 April 2013
Published: 10 May 2013
Email:
Bing Xu* - bxu@brandeis.edu
* Corresponding author
Associate Editor: N. Sewald
Keywords:
© 2013 Li et al; licensee Beilstein-Institut.
License and terms: see end of document.
self-delivery; hydrogel; multifunctional; nanofibers; NSAID;
self-assembly; supramolecular; topical use
Abstract
Here we report supramolecular hydrogelators made of nonsteroidal anti-inflammatory drugs (NSAID) and small peptides. The
covalent linkage of Phe–Phe and NSAIDs results in conjugates that self-assemble in water to form molecular nanofibers as the
matrices of hydrogels. When the NSAID is naproxen (1), the resultant hydrogelator 1a forms a hydrogel at a critical concentration
(cgc) of 0.2 wt % at pH 7.0. Hydrogelator 1a, also acting as a general motif, enables enzymatic hydrogelation in which the
precursor turns into a hydrogelator upon hydrolysis catalyzed by a phosphatase at physiological conditions. The conjugates of
Phe–Phe with other NSAIDs, such as (R)-flurbiprofen (2), racemic flurbiprofen (3), and racemic ibuprofen (4), are able to form
molecular hydrogels, except in the case of aspirin (5). After the conjugation with the small peptides, NSAIDs exhibit improved
selectivity to their targets. In addition, the peptides made of D-amino acids help preserve the activities of NSAIDs. Besides demon-
strating that common NSAIDs are excellent candidates to promote aromatic–aromatic interaction in water to form hydrogels, this
work contributes to the development of functional molecules that have dual or multiple roles and ultimately may lead to new molec-
ular hydrogels of therapeutic agents for topical use.
Introduction
This article reports the design, synthesis, and characterization of tional supramolecular hydrogels. Sharing similar features with
hydrogelators made of non-steroidal anti-inflammatory drugs the extracellular matrices of tissues (e.g., consisting of three-
(NSAID) and small peptides for the development of multifunc- dimensional networks and a significant amount of water),
908

Beilstein J. Org. Chem. 2013, 9, 908–917.
hydrogels have become an attractive choice for developing the molecular hydrogels of NSAIDs because topical use of the
biomaterials for a variety of applications. For example, hydro- hydrogels of NSAIDs is an attractive therapeutic option for
gels have frequently served as scaffolds for tissue engineering treating acute pain [49].
and as carriers for drug delivery [1-4]. In the applications of
drug delivery, it is common to use hydrogels made of Because of their analgesic, antipyretic, and anti-inflammatory
biodegradable polymers to encapsulate therapeutic agents for effects (when being used in high dosage), NSAIDs are widely
controlled release of drugs by adjusting the pore sizes and func- used for the treatment of acute or chronic pain or inflamma-
tionality of the polymer networks [5,6]. Despite the tremendous tions. Despite the widespread systemic use of NSAIDs, the
success of this approach, several inherent shortcomings still adverse gastrointestinal and renal effects and cardiovascular
limit the application of polymeric hydrogels. For example, the risks associated with NSAIDs have led to the development of
degradation of the polymers usually generates acidic monomers, topical NSAIDs formulations, such as patch, gel, and solution
which, sometimes, cause undesired inflammatory responses [7]. [50-52]. Encouraged by the successful FDA approval of
In most of the cases, the polymers themselves are passive, diclofenac gel [53] and several reports of small molecule hydro-
which unavoidably results in the limited loading of drug mole- gels formed by NSAID derivatives [48,54-56] we intend to
cules [8,9]. Though possible, it is relatively difficult to function- explore supramolecular hydrogels of other NSAIDs. Specifi-
alize the polymers with drug molecules. Even though it has cally, we use naproxen (denoted as Npx in this report), an over-
been attempted, the release of the drugs from the polymer back- the-counter NSAID, to generate a new hydrogelator 1a that only
bone remains a nontrivial issue. Despite being constantly im- consists of naproxen and diphenylalanine (FF). We use
proved, these limitations call for the exploration and develop- diphenylalanine because it is able to self-assemble to form
ment of new functional biomaterials and therapeutics.
nanotubes [57]. We find that compound 1a, forming a hydrogel
at the critical gelation concentration (cgc) of 0.2 wt % at pH
The recent development of supramolecular nanofibers and 7.0, also serves as a general motif to enable enzymatic hydroge-
hydrogels [10-25] presents an exciting opportunity for devel- lation that converts 1c to 1d by a phosphatase and results in a
oping new types of biomaterials [26-29]. While the early works hydrogel of 1d under physiological conditions. In addition to
have centered on the use of nanofibers of oligopeptides to form naproxen, we evaluate the abilities of other NSAIDs (Scheme 1)
hydrogels as a passive scaffold for tissue engineering and drug as building blocks of hydrogelators and find that the conjugates
delivery [30], the seminal work by Stupp et al., in which func- of FF and (R)-flurbiprofen (2), racemic flurbiprofen (3) or
tional hydrogels based on supramolecular nanofibers regulate racemic ibuprofen (4) form supramolecular hydrogels. More-
the differentiation of neural progenitor cells [31], has demon- over, our previous examination of the efficacy of the hydrogela-
strated undoubtedly the power and promises of the rational tors derived from naproxen shows that the conjugation of small
incorporation of functional motifs into the building blocks of peptides may improve the selectivity of naproxen for inhibiting
the supramolecular nanofibers and hydrogels. Besides being COX-2 and thus reduces their adverse effect [58]. Although the
used for three-dimensional cell cultures [32], supramolecular inhibition efficacies of the hydrogelators for both enzymes
hydrogels also find applications in typing [33] and against COX-1 and COX-2 are lower than naproxen, the use of
bacteria [34-38], and in enzyme assays [23,39], catalysis [40], D-peptide for conjugation helps preserve the activities of
and inhibiting cancer cells [41-44]. These successful examples naproxen. These results provide useful insights for the optimi-
further underscore the potentials of functional supramolecular zation of these hydrogelators. Besides serving as a potential
hydrogels as new and useful biomaterials.
alternative approach to polymeric hydrogels [59,60], this work
contributes to the development of bioactive molecules that have
Since the formation of supramolecular hydrogels relies on the dual or multiple roles, for example, as therapeutic agents and
small molecules (i.e., the hydrogelators) that self-assemble in delivery carriers [61,62].
water through noncovalent interactions [45,46], these hydro-
Results and Discussion
gels are inherently easily biodegradable. To meet the prerequi-
sites for self-delivery systems, besides biodegradability, the Synthesis
hydrogelators should preserve the pharmacological efficacy of Scheme 2 shows the typical synthetic route of the conjugates of
the therapeutic building blocks, any side effects should be mini- NSAID and small peptides exemplified in the case of naproxen.
mized and the biocompatibility maximized. Therefore, a con- In this process, naproxen (1) first reacts with N-hydroxysuccin-
venient starting point for developing the molecular hydrogels of imide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC) in
therapeutic agents is to construct hydrogelators only consisting chloroform to afford the NHS ester of 1, which reacts with a
of the therapeutic agent and biocompatible motifs [47,48]. phenylalanine in the mixed solvent of acetone and water to
Among a large pool of therapeutic agents, we choose to develop produce 11. The same coupling procedure allows the covalent
909

Beilstein J. Org. Chem. 2013, 9, 908–917.
Scheme 1: The structures of the NSAIDs and peptides explored as the building blocks of hydrogelators in this work.
attachment of second phenylalanine on the C-terminal of 11 to to afford compounds (R)-Fbp-FF (2a), (R)-Fbp-ff (2b), and
give 1a. Being activated by NHS, 1a reacts with phosphotyro- (RS)-Fbp-FF (3a) (Fbp = flurbiprofen). The attachment of
sine (pY) to yield 1c after purification by HPLC. Using the racemic ibuprofen to L-diphenylalanine yields compound (RS)-
same procedure, we connect naproxen to D-peptides to make Ibp-FF (4a) (Ibp = ibuprofen). Since the direct use of acetylsali-
Npxff (1b), Npxffy(p) (1e), and Npxffy (1f), because D-amino cylic acid (aspirin) to connect with dipeptides results in a com-
acid based hydrogelators are able to resist proteases [63].
pound hydrolyzing easily at the ester bond of aspirin, we use
salicylic acid (Sal) instead to conjugate with diphenylalanine
Scheme 2 shows the general synthetic route of other NSAID and dialanine (AA) to make compounds 5a and 5g, respective-
containing hydrogelators and lists all the NSAID-based conju- ly.
gates studied in this work. Generally, after reacting with NHS
and DCC in chloroform, the NSAID becomes a NHS ester, Self-assembly and hydrogelation
which reacts with the small peptides (e.g., Phe–Phe, After the synthesis of the NSAID/small-peptid conjugates, we
Phe–Phe–Tyr, or Ala–Ala) in the mixed solvent of acetone and examined the capability of the conjugates to self-assemble in
water. We have produced these NSAID-containing hydrogela- water to form supramolecular nanofibers and hydrogels
tors with yields from 40% to 60% after purification by HPLC or (Table 1). Typically, the addition of 8.0 mg of Npx-FF (1a) into
column chromatography. For example, flurbiprofen (R-enan- 1.0 mL of water under basic conditions (pH 9.0) affords a solu-
tiomer 2 or racemic 3) conjugates with L- or D-diphenylalanine tion, which turns into a suspension at pH 7.0. Upon heating to
910

Beilstein J. Org. Chem. 2013, 9, 908–917.
Scheme 2: The synthetic route of the naproxen-containing hydrogelators and the corresponding precursors: (i) NHS, DCC, CHCl3, 2 h; Phe, pH 8.5,
12 h. (ii) NHS, DCC, CHCl3, 12 h; Tyr(p), pH 8.5, 24 h. (iii) alkaline phosphatase, pH 7.6.
60 °C, the suspension turns to a clear solution, and then The hydrogelation of 1a also offers an opportunity to examine
becomes a transparent hydrogel after being cooled to room whether the attachment of a tyrosine phosphate residue to 1a
temperature (Figure 1A). The treatment with a small amount of allows enzymatic hydrogelation. As a soluble precursor, 1c
base prior to the adjustment of pH appeared to be a necessary (15.0 mg) dissolves in water (1.0 mL) at pH 7.6. After being
step for making the hydrogel. Being thermally reversible and treated with alkaline phosphatase (5.0 U/mL), the solution of 1c
relatively sensitive to the temperature, the hydrogel of 1a quickly transforms to a clear hydrogel within one hour. Due to
exhibits excellent recovery properties (Supporting Information the additional tyrosine residue, 1d, however, exhibits much
File 1, Figure S4) [64].
better solubility in water than does 1a. Thus, 1d remains as a
911

Beilstein J. Org. Chem. 2013, 9, 908–917.
Table 1: The properties of the NSAID-containing hydrogels.
concentration
(wt %)
method of
preparation
pH
components
gel properties
transparent gel, thermal reversible, mechanical
recovery
1a
0.8
7.0 heat–cool
4.0 acidic pH
7.6 enzyme
naproxen, L-Phe–L-Phe
naproxen, D-Phe–D-Phe
1b 0.8
1d 1.5
opaque hydrogel, precipitate at high T,
naproxen,
L-Phe–L-Phe–L-Tyr
transparent gel, mechanical recovery
naproxen,
D-Phe–D-Phe–D-Tyr
1f
0.8
0.8
7.6 enzyme
transparent gel
(R)-flurbiprofen,
L-Phe–L-Phe
slightly opaque hydrogel, thermal reversible,
weak mechanical recovery
2a
7.2 heat–cool
7.2 heat–cool
7.2 heat–cool
(R)-flurbiprofen,
D-Phe–D-Phe
semitransparent hydrogel, thermal reversible,
weak mechanical recovery
2b 0.8
(RS)-flurbiprofen,
L-Phe–L-Phe
semitransparent hydrogel, thermal reversible,
weak mechanical recovery
3a
4a
0.8
0.8
(RS)-ibuprofen,
L-Phe–L-Phe
semitransparent hydrogel, thermal reversible,
weak mechanical recovery
7.2 heat–cool
4.0 acidic pH
1.0 acidic pH
1g 0.8
3g 0.8
naproxen, L-Ala–L-Ala
opaque hydrogel, poor stability at rt
(RS)-flurbiprofen,
L-Ala–L-Ala
opaque hydrogel, poor stability at rt
Figure 1: Optical images of the hydrogels formed by (A) 1a (0.8 wt %, pH 7.0); (B) 1b (0.8 wt %, pH 4.0); (C) 1d (1.5 wt %, pH 7.6), obtained by
treating 1c with alkaline phosphatase (5.0 U/mL); (D) 1f (0.8 wt %, pH 7.6), obtained by treating 1e with alkaline phosphatase (5.0 U/mL); (E) 2a
(0.8 wt %, pH 7.2); (F) 2b (0.8 wt %, pH 7.2); (G) 3a (0.8 wt %, pH 7.2); (H) 4a (0.8 wt %, pH 7.2); (I) 1g (0.8 wt %, pH 4.0); (J) 3g (0.8 wt %, pH 1.0).
solution at the concentration of 0.8 wt %, and only forms a hydrogel suggests that 1b tends to form microcrystals [65-68]
stable hydrogel (Figure 1C) when the concentration of 1d that constitute the weakly cross-linked matrices of the hydrogel.
reaches 1.5 wt %. As in the case of 1a, the hydrogel of 1d also The addition of a D-tyrosine phosphate residue to 1b affords the
shows good recovery properties.
precursor 1e. Despite containing a D-tyrosine phosphate, 1e
(0.8 wt %, pH 7.6) undergoes dephosphorylation in the pres-
Being the diastereomer of 1a, hydrogelator 1b exhibits quite ence of alkaline phosphatase (5.0 U/mL) to form the hydroge-
different behavior in terms of self-assembly and hydrogelation. lator 1f, which results in a transparent hydrogel (Figure 1D).
For example, at a concentration of 0.8 wt %, 1b forms a solu- Unlike 1d, only 0.8 wt % of 1f is necessary to form the
tion at pH 9.0 and 60 °C, and the solution remains after the hydrogel at pH 7.6.
conditions have been adjusted to pH 7.0 and room temperature.
Further decreasing of the pH value of the solution of 1b to 4.0 As a hydrogelator, 2a displays different characteristics to those
results in an opaque hydrogel (Figure 1B), which lacks thermal of 2b. 2a (8.0 mg) dissolves in water (1.0 mL) at pH 9 when
reversibility and turns into white precipitates when it is heated heated to 85 °C. The clear solution of 2a turns into a stable,
to about 60 °C. Additionally, the white and opaque color of the thermally reversible, and slightly opaque hydrogel (Figure 1E)
912

Beilstein J. Org. Chem. 2013, 9, 908–917.
after being stored at room temperature for 12 h at pH 7.2. 2b morphology from that of 1a, and likely contributes to the
exhibits a better solubility than 2a because 0.8 wt % of 2b gives thermal irreversibility of the hydrogel of 1b. Besides, the low
a clear solution at pH 9.0 when being heating to 65 °C. Simi- density of the network in hydrogel 1b (Figure S1, Supporting
larly, the solution of 2b turns into a semitransparent hydrogel Information File 1) [64] agrees with its poor reversibility. As
(Figure 1F) after the pH is decreased to 7.2. 2b forms thermally shown in Figure 2C and Figure 2D, both the hydrogels of 1d
reversible hydrogel within 2 min after its solution is cooled to and 1f comprise uniform long nanofibers, whose average widths
room temperature from 65 °C, which is much faster than the are 6–7 nm. The exceptional high density of nanofibers in the
hydrogelation of 2a. The commercially available flurbiprofen is hydrogel of 1d likely originates from the relatively high
the racemic mixture 3. Similar to the behavior of 2a, 3a concentration of 1d in the hydrogel.
(8.0 mg) dissolves in water (1.0 mL) at pH 9.0 and 75 °C, and
the solution of 3a becomes a stable semitransparent hydrogel
(Figure 1G) within 5 min at room temperature after the pH of
the solution is adjusted to 7.2. Although being thermally revers-
ible, the hydrogel of 3a exhibits poor recovery: the hydrogel of
3a takes more than 24 hours to recover from the sol–gel state
after being disrupted by an external force. Being made from a
racemic mixture of ibuprofen (4), 4a (8.0 mg) dissolves in water
(1.0 mL) at pH 9 and 75 °C, and the solution of 4a becomes a
stable semitransparent hydrogel (Figure 1H) after standing at
room temperature and pH 7.2 overnight. Despite its thermal re-
versibility, 4a shows weak recovery properties. Compound 5a
fails to form a hydrogel, likely due to the insufficient
aromatic–aromatic interaction originating from the salicylic
acid groups. In order to confirm that the aromatic–aromatic
interactions originating from Phe–Phe are essential for the self-
assembly of the hydrogelators of NSAIDs to form the
nanofibers as the matrices of the hydrogels, we studied the gela-
tion properties of the conjugates of NSAIDs and Ala–Ala, a
nonaromatic dipeptide. The conjugates of NSAIDs and
Ala–Ala, 1g, 3g, 4g, and 5g, behave differently from those
conjugates that contain Phe–Phe. At a concentration of
0.8 wt %, while 1g and 3g afford white, opaque hydrogels at pH
4.0 and 1.0, respectively, compounds 4g and 5g fail to form a
hydrogel. The hydrogels of 1g and 3g shrink within 30 min after
hydrogelation and tend to precipitate at room temperature over
24 h, indicating poor stability of the hydrogels of 1g and
3g. This result further confirms the critical role of
aromatic–aromatic interaction for stabilizing molecular self-
assembly in water.
Transmission electron microscopy (TEM)
In addition to the macroscopic phase transition such as hydroge-
lation, another hallmark of molecular self-assembly in water is
the formation of ordered nanostructures (e.g., nanofibers or
nanoparticles). As revealed by the TEM images (Figure 2), the
hydrogel of 1a comprises long nanofibers that entangle to form
Figure 2: The TEM images of the matrices of the hydrogels formed by
the network. The widths of nanofibers in the gel of 1a appear to
be nonuniform, displaying minimum width at 6 nm and
maximum width at 20 nm (Figure 2A). The TEM image of the
hydrogel of 1b shows helical and rigid nanofibers with an
average width of around 48 nm (Figure 2B), which is a different
(A) 1a (0.8 wt %, pH 7.0); (B) 1b (0.8 wt %, pH 4.0); (C) 1d (1.5 wt %,
pH 7.6) formed by treating 1c with alkaline phosphatase (5.0 U/mL);
(D) 1f (0.8 wt %, pH 7.6) formed by treating 1e with alkaline phos-
phatase (5.0 U/mL); (E) 2a (0.8 wt %, pH 7.2); (F) 2b (0.8 wt %, pH
7.2); (G) 3a (0.8 wt %, pH 7.2); (H) 4a (0.8 wt %, pH 7.2); (I) 1g
(0.8 wt %, pH 4); (J) 3g (0.8 wt %, pH 1.0). The scale bar is 100 nm.
913

Beilstein J. Org. Chem. 2013, 9, 908–917.
The hydrogel of 2a comprises two kinds of nanostructures, i.e., frequencies and different oscillation strains, exhibit a strain-
nanofibers and nanoparticles, visible in the TEM images. As independent storage modulus (G') until reaching their critical
shown in Figure 2E, long nanofibers with an average width of strain, at which the values of G′ start to decrease drastically due
17 nm entangle together to form larger helical nanofibers with to the breakdown of the networks of the hydrogels. In addition
an average width of 30 nm. Higher-magnification TEM shows to the critical strain, the dynamic strain sweep provides the
that the large helical fibers comprise fibrils of 17 nm in width maximum G' values in the linear region. The naproxen-
(Figure S1, Supporting Information File 1) [64]. In addition to containing hydrogels show maximum storage moduli from
the presence of the nanofibers, the nanoparticles (10–20 nm in 4.0 × 102 to 4.9 × 104 Pa (Table 2). The hydrogel of 1a has a
diameter) also exist in the hydrogel of 2a. Different from the maximum G' of 2.1 × 103 Pa and critical strain of 0.62%, and
hydrogel of 2a, hydrogel 2b comprises long, uniform nanofibers the hydrogel of 1b has the highest maximum G' (4.9 × 104 Pa),
with an average width of 12 nm (Figure 2F). Interestingly, the agreeing with the well cross-linked network in the hydrogel of
TEM of the hydrogel of 3a presents similar nanostructures to 1b resulted from the long and wide (48 nm) nanofibers. Since
those in the hydrogel of 2b. As shown in Figure 2G, the long the tyrosine residue renders 1d and 1f with better solubility than
uniform nanofibers in the hydrogel of 3a have an average width for 1a and 1b (Table S1, Supporting Information File 1) [64],
of 12 nm. Higher magnification of the TEM of the hydrogel of the hydrogels of 1d and 1f have smaller maximum storage
3a indicates that the nanofibers of 3a consist of two nanofibrils moduli (6.2 × 102 and 4.0 × 102 Pa) than do the hydrogels of 1a
with an average width of 6 nm (Figure S1, Supporting Informa- and 1b. The thin and flexible nanofibers in the hydrogels of 1d
tion File 1) [64]. As shown in Figure 2H, the hydrogel of 4a and 1f also agree with their rheological properties.
also consists of uniform nanofibers with an average width of
13 nm. These nanofibers arrange into a network that has very (R)-Flurbiprofen-containing hydrogelators 2a and 2b exhibit
low density. While the TEM image (Figure 2I) of the hydrogel maximum storage moduli of 1.5 × 102 and 8.7 × 102 Pa, res-
of 1g clearly shows nanocrystals with a length over 450 nm and pectively, which are smaller than those of the hydrogels of 1a
width varying from 35 nm to 200 nm, the TEM image and 1b. Such a difference is consistent with the less
(Figure 2J) of the hydrogel of 3g exhibits uniform nanotubes aromatic–aromatic interaction provided by the flurbiprofen
that have an outer width of 22 nm and inner width of 8 nm. motif due to its smaller conjugate system compared with the
These nanotubes form a poorly cross-linked network, which naproxen group in 1a and 1b. The maximum G' of the hydrogel
agrees with the instability of the hydrogel of 3g.
of 2b (8.9 × 102 Pa) is larger than that of the hydrogel of 2a
(1.5 × 102 Pa), agreeing with the higher density of the
nanofibers in the hydrogel of 2b. Interestingly, the gel of 3a,
Rheology
Since viscoelasticity is an essential feature of hydrogels [69], which comprises 2a and (S)-flurbiprofen-FF, exhibits a higher
we use rheometry to characterize the hydrogels. Based on the maximum G' (1.3 × 103 Pa) in strain sweep and larger critical
stability of these hydrogels, we choose to study the rheological strain (3.7%) than those values of 2a or 2b, suggesting that the
properties of the hydrogels of 1a, 1b, 1d, 1f, 2a, 2b, 3a and 4a, combination of 2a and (S)-flurbiprofen-FF affords a more stable
and list their rheology data in Table 2. During the dynamic hydrogel. This result agrees with the TEM image of 3a, which
strain sweep, all these hydrogels, under constant oscillation exhibits the highest density of its network among the flur-
Table 2: The rheological properties and TEM characteristics of the NSAID-containing hydrogels.
cgc
(wt %)
max. G' a
(Pa)
critical straina
(%)
G' b
(Pa)
nanofiber widthc
(nm)
compound
1a
1b
1dd
1f
0.2
0.2
1.0
0.2
0.2
0.2
0.2
0.3
2100
49000
620
0.62
1.0
1700
53000
610
6–20
48
6.5
7
0.39
1.6
400
620
2a
2b
3a
4a
150
0.96
1.8
150
17
12
6
890
990
1300
7.8
3.7
1500
13
0.57
13
aDynamic strain sweep; bDynamic frequency sweep, the value is taken at a frequency equal to 6.28 rad/s; cAverage; dThe concentration of hydrogel
1d is 1.5 wt %, while the others are 0.8 wt %.
914

Beilstein J. Org. Chem. 2013, 9, 908–917.
biprofen-containing hydrogels. Unlike the hydrogels containing 1a and 1d, like other supramolecular gels [70,71], are able to
naproxen or flurbiprofen, ibuprofen-containing hydrogel 4a re-establish networks rapidly after deformation.
behaves as a relatively weak hydrogel and exhibits a maximum
G' of 7.8 Pa and critical strain 0.57%, clearly indicating that the
isopropyl-substituted phenyl group is less efficient at providing
aromatic–aromatic interaction for hydrogelation than naproxen
or flubiprofen group are.
After obtaining the critical strains of the hydrogels, we measure
the frequency dependence of their storage and loss moduli using
dynamic frequency sweep at constant oscillation amplitude and
temperature but varying oscillation frequency (0.1–200 rad/s).
All of these hydrogels exhibit little dependence on frequency
(Figure S3, Supporting Information File 1) [64], suggesting that
the matrices of gels have good tolerance to the external shear
force. In addition, the storage moduli of all these hydrogels in
the frequency sweep are comparable with storage moduli of the
constant region in the strain sweep. Hydrogel 1b has the highest
G' (5.3 × 104 Pa), while hydrogels 1d and 1f appear to have the
lowest two G' values (6.1 × 102 Pa and 6.2 × 102 Pa) among the
naproxen-containing hydrogels. Hydrogels of 2a and 2b have
smaller G' values, 1.5 × 102 Pa and 9.9 × 102 Pa, respectively,
Figure 3: Recovery of the storage moduli of the gels formed by
0.8 wt % of 1a at pH 7.0 and 1.5 wt % of 1d formed by using alkaline
phosphatase (5.0 U/mL) to treat the solution of 1c at pH 7.6.
than those of hydrogels 1a and 1b, which likely originate from Cytotoxicity
their structure difference. Hydrogel 3a has a G' value of To evaluate the biocompatibility of these NSAID hydrogelators,
1.5 × 103 Pa, which is larger than that of hydrogel of 4a (13 Pa), we select 1a, 1c, and 1d to test their cytotoxicity by incubating
partially due to more effective aromatic–aromatic interaction of them with HeLa cells for 72 h at 37 °C. As shown in Figure 4,
the flurbiprofen groups compared to the ibuprofen.
1a, 1c, and 1d exhibit IC50 values of 206 µM, 321 µM, and
294 µM, respectively. Among these three hydrogels, 1a lacks
Both hydrogels of 1a and 1d possess excellent thermal recovery the tyrosine residue and is less soluble in water, thus exhibiting
properties (Figure S4, Supporting Information File 1) [64], indi- relatively low IC50. All of 1a, 1c and 1d, exhibit IC50 values
cating that these molecules self-assemble to re-establish the larger than 200 µM, suggesting that they are relatively biocom-
network rapidly after deformation caused by a perturbation. To patible.
characterize the recovery properties of the gels of 1a and 1d, we
first measure the G' values of the gels at 0.4% strain for 10 min
to obtain their original storage moduli, and then apply a large-
amplitude oscillation with 100% strain and 6.28 rad/s angular
frequency to perturb the structure of the hydrogels for 10 min.
After removing this large amplitude of oscillation, we immedi-
ately measure the storage moduli of the gels at 0.4% of strain,
and then calculate their recovery percentages by dividing them
by their original moduli. As shown in Figure 3, G' of hydrogel
1a decreases to 0.5% of its original storage modulus when
being exposed to the large-amplitude oscillation (100% of
strain). After the removal of the applied large oscillation, the G'
value of hydrogel 1a immediately recovers by 50% and reaches
100% recovery in less than 5 min. Similarly, hydrogel 1d also
exhibits a fairly fast recovery of G'. The application of the large
oscillation for 10 min decreases the G' of hydrogel 1d to 2.5%
of its original storage modulus. As soon as the external large
Figure 4: The IC50 values of 1a, 1c and 1d incubated with HeLa cells
after 72 h.
oscillation is stopped, hydrogel 1d recovers by 25% and reaches
100% recovery after 20 min. These data suggest that hydrogels
915

Beilstein J. Org. Chem. 2013, 9, 908–917.
11.Banerjee, S.; Das, R. K.; Maitra, U. J. Mater. Chem. 2009, 19, 6649.
doi:10.1039/b819218a
Conclusion
In conclusion, we have systematically investigated a new type
of supramolecular hydrogelator made of nonsteroidal anti-
inflammatory drugs (NSAIDs). The direct conjugation of
NSAIDs and small peptides (Phe–Phe) generates molecules that
self-assemble in water to form hydrogels. The large difference
in gelation properties by changing small peptides from
Phe–Phe to Ala–Ala confirms the important role of the
aromatic–aromatic interaction between diphenylalanine groups
[24]. In addition, the molecular structures of NSAIDs also
affect their gelation stability. As a useful member in NSAIDs,
naproxen conjugates with Phe–Phe to give a promising hydro-
gelator (NpxFF) that may act as a general motif to enable
hydrogelation of other bioactive molecules. By providing useful
insight for design of the NSAID hydrogelators, this approach
contributes to the development of bioactive molecules that have
dual or multiple roles, such as hydrogelators and therapeutic
agents.
12.Adhikari, B.; Palui, G.; Banerjee, A. Soft Matter 2009, 5, 3452.
doi:10.1039/b905985g
13.Adams, D. J.; Topham, P. D. Soft Matter 2010, 6, 3707.
doi:10.1039/c000813c
14.Chen, J.; McNeil, A. J. J. Am. Chem. Soc. 2008, 130, 16496.
doi:10.1021/ja807651a
15.Dastidar, P. Chem. Soc. Rev. 2008, 37, 2699. doi:10.1039/b807346e
16.King, K. N.; McNeil, A. J. Chem. Commun. 2010, 46, 3511.
doi:10.1039/c002081h
17.Qiu, Z.; Yu, H.; Li, J.; Wang, Y.; Zhang, Y. Chem. Commun. 2009,
3342. doi:10.1039/b822840j
18.Hirst, A. R.; Coates, I. A.; Boucheteau, T. R.; Miravet, J. F.;
Escuder, B.; Castelletto, V.; Hamley, I. W.; Smith, D. K.
J. Am. Chem. Soc. 2008, 130, 9113. doi:10.1021/ja801804c
19.Kameta, N.; Yoshida, K.; Masuda, M.; Shimizu, T. Chem. Mater. 2009,
21, 5892. doi:10.1021/cm903108h
20.Liao, S. W.; Yu, T.-B.; Guan, Z. J. Am. Chem. Soc. 2009, 131, 17638.
doi:10.1021/ja907097t
21.Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A. K.;
Ulijn, R. V. Nat. Nanotechnol. 2009, 4, 19. doi:10.1038/nnano.2008.378
22.Wu, Z. L.; Kurokawa, T.; Liang, S.; Furukawa, H.; Gong, J. P.
J. Am. Chem. Soc. 2010, 132, 10064. doi:10.1021/ja101969k
23.Yang, Z.; Liang, G.; Xu, B. Acc. Chem. Res. 2008, 41, 315.
doi:10.1021/ar7001914
Supporting Information
Supporting Information File 1
Experimental details.
24.Zhang, Y.; Kuang, Y.; Gao, Y.; Xu, B. Langmuir 2011, 27, 529.
doi:10.1021/la1020324
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-104-S1.pdf]
25.Leong, W. L.; Tam, A. Y.-Y.; Batabyal, S. K.; Koh, L. W.; Kasapis, S.;
Yam, V. W.-W.; Vittal, J. J. Chem. Commun. 2008, 3628.
doi:10.1039/b807478j
26.Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.;
Kretsinger, J. J. Am. Chem. Soc. 2002, 124, 15030.
doi:10.1021/ja027993g
Acknowledgements
This work is supported by the Human Frontier Program (HFSP,
RGP 0056/2008), NIH (R01CA142746) and a start-up fund
from Brandeis University and assisted by the Brandeis Univer-
sity EM Facility.
27.Collier, J. H.; Segura, T. Biomaterials 2011, 32, 4198.
doi:10.1016/j.biomaterials.2011.02.030
28.Lovell, J. F.; Roxin, A.; Ng, K. K.; Qi, Q.; McMullen, J. D.;
DaCosta, R. S.; Zheng, G. Biomacromolecules 2011, 12, 3115.
doi:10.1021/bm200784s
29.Kumar, K. K.; Elango, M.; Subramanian, V.; Das, T. M. New J. Chem.
2009, 33, 1570. doi:10.1039/b821126d
References
1. Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869.
30.Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A.
Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 3334.
doi:10.1021/cr000108x
2. Karp, J. M.; Langer, R. Curr. Opin. Biotechnol. 2007, 18, 454.
doi:10.1016/j.copbio.2007.09.008
doi:10.1073/pnas.90.8.3334
31.Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.;
Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352.
doi:10.1126/science.1093783
3. Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47.
doi:10.1038/nbt1055
4. Takashima, Y.; Yuting, Y.; Otsubo, M.; Yamaguchi, H.; Harada, A.
Beilstein J. Org. Chem. 2012, 8, 1594. doi:10.3762/bjoc.8.182
5. Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337.
doi:10.1016/S0142-9612(03)00340-5
32.Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. F.; Saiani, A.;
Gough, J. E.; Ulijn, R. V. Adv. Mater. 2006, 18, 611.
doi:10.1002/adma.200501522
33.Yang, Z.; Ho, P.-L.; Liang, G.; Chow, K. H.; Wang, Q.; Cao, Y.; Guo, Z.;
Xu, B. J. Am. Chem. Soc. 2007, 129, 266. doi:10.1021/ja0675604
34.Xing, B.; Yu, C.-W.; Chow, K.-H.; Ho, P.-L.; Fu, D.; Xu, B.
J. Am. Chem. Soc. 2002, 124, 14846. doi:10.1021/ja028539f
35.Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P.
J. Am. Chem. Soc. 2007, 129, 14793. doi:10.1021/ja076300z
36.Yang, Z.; Liang, G.; Guo, Z.; Guo, Z.; Xu, B. Angew. Chem., Int. Ed.
2007, 46, 8216. doi:10.1002/anie.200701697
6. Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M.
Chem. Rev. 1999, 99, 3181. doi:10.1021/cr940351u
7. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater.
2006, 18, 1345. doi:10.1002/adma.200501612
8. Torchilin, V. P. J. Controlled Release 2001, 73, 137.
doi:10.1016/S0168-3659(01)00299-1
9. Brazel, C. S.; Peppas, N. A. Polymer 1999, 40, 3383.
doi:10.1016/S0032-3861(98)00546-1
37.Zhao, F.; Ma, M. L.; Xu, B. Chem. Soc. Rev. 2009, 38, 883.
doi:10.1039/b806410p
10.Estroff, L. A.; Hamilton, A. D. Chem. Rev. 2004, 104, 1201.
doi:10.1021/cr0302049
916

Beilstein J. Org. Chem. 2013, 9, 908–917.
38.Brahmachari, S.; Debnath, S.; Dutta, S.; Das, P. K.
Beilstein J. Org. Chem. 2010, 6, 859. doi:10.3762/bjoc.6.101
39.Yang, Z. M.; Xu, B. Chem. Commun. 2004, 2424.
doi:10.1039/b408897b
65.Terech, P.; Weiss, R. G. Chem. Rev. 1997, 97, 3133.
doi:10.1021/cr9700282
66.Kar, T.; Debnath, S.; Das, D.; Shome, A.; Das, P. K. Langmuir 2009,
25, 8639. doi:10.1021/la804235e
40.Wang, Q.; Yang, Z.; Zhang, X.; Xiao, X.; Chang, C. K.; Xu, B.
Angew. Chem., Int. Ed. 2007, 46, 4285. doi:10.1002/anie.200700404
41.Gao, Y.; Kuang, Y.; Guo, Z.-F.; Guo, Z.; Krauss, I. J.; Xu, B.
J. Am. Chem. Soc. 2009, 131, 13576. doi:10.1021/ja904411z
42.Bouhadir, K. H.; Alsberg, E.; Mooney, D. J. Biomaterials 2001, 22,
2625. doi:10.1016/S0142-9612(01)00003-5
67.George, M.; Funkhouser, G. P.; Weiss, R. G. Langmuir 2008, 24, 3537.
doi:10.1021/la703556d
68.Ostuni, E.; Kamaras, P.; Weiss, R. G. Angew. Chem., Int. Ed. Engl.
1996, 35, 1324. doi:10.1002/anie.199613241
69.Yan, C.; Pochan, D. J. Chem. Soc. Rev. 2010, 39, 3528.
doi:10.1039/b919449p
43.Standley, S. M.; Toft, D. J.; Cheng, H.; Soukasene, S.; Chen, J.;
Raja, S. M.; Band, V.; Band, H.; Cryns, V. L.; Stupp, S. I. Cancer Res.
2010, 70, 3020. doi:10.1158/0008-5472.CAN-09-3267
44.Yang, Z. M.; Xu, K. M.; Guo, Z. F.; Guo, Z. H.; Xu, B. Adv. Mater. 2007,
19, 3152. doi:10.1002/adma.200701971
70.Adams, D. J.; Mullen, L. M.; Berta, M.; Chen, L.; Frith, W. J. Soft Matter
2010, 6, 1971. doi:10.1039/b921863g
71.Mukhopadhyay, S.; Maitra, U.; Ira, K.; Krishnamoorthy, G.; Schmidt, J.;
Talmon, Y. J. Am. Chem. Soc. 2004, 126, 15905.
doi:10.1021/ja046788t
45.Shi, J.; Gao, Y.; Yang, Z.; Xu, B. Beilstein J. Org. Chem. 2011, 7, 167.
doi:10.3762/bjoc.7.23
46.Pasc, A.; Akong, F. O.; Cosgun, S.; Gérardin, C.
Beilstein J. Org. Chem. 2010, 6, 973. doi:10.3762/bjoc.6.109
47.Vemula, P. K.; Li, J.; John, G. J. Am. Chem. Soc. 2006, 128, 8932.
doi:10.1021/ja062650u
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
48.Bhuniya, S.; Seo, Y. J.; Kim, B. H. Tetrahedron Lett. 2006, 47, 7153.
doi:10.1016/j.tetlet.2006.08.002
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
49.Moore, R. A.; Tramèr, M. R.; Carroll, D.; Wiffen, P. J.; McQuay, H. J.
BMJ 1998, 316, 333. doi:10.1136/bmj.316.7128.333
50.Cevc, G.; Mazgareanu, S.; Rother, M.; Vierl, U. Int. J. Pharm. 2008,
359, 190. doi:10.1016/j.ijpharm.2008.04.005
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
51.Lorenzo-Lamosa, M. L.; Remuñán-López, C.; Vila-Jato, J. L.;
Alonso, M. J. J. Controlled Release 1998, 52, 109.
doi:10.1016/S0168-3659(97)00203-4
(http://www.beilstein-journals.org/bjoc)
52.Csoka, G.; Racz, I.; Marton, S.; Balogh, E.; Farkas, E. Pharm. Ind.
1999, 61, 88.
The definitive version of this article is the electronic one
which can be found at:
53.Banning, M. Expert Opin. Pharmacother. 2008, 9, 2921.
doi:10.1517/14656566.9.16.2921
doi:10.3762/bjoc.9.104
54.Park, S. M.; Lee, Y. S.; Kim, B. H. Chem. Commun. 2003, 2912.
doi:10.1039/b311249g
55.Foster, J. A.; Piepenbrock, M.-O. M.; Lloyd, G. O.; Clarke, N.;
Howard, J. A. K.; Steed, J. W. Nat. Chem. 2010, 2, 1037.
doi:10.1038/nchem.859
56.Bhuniya, S.; Park, S. M.; Kim, B. H. Org. Lett. 2005, 7, 1741.
doi:10.1021/ol050300r
57.Görbitz, C. H. Chem.–Eur. J. 2001, 7, 5153.
doi:10.1002/1521-3765(20011203)7:23<5153::AID-CHEM5153>3.0.C
O;2-N
58.Li, J.; Kuang, Y.; Gao, Y.; Du, X.; Shi, J.; Xu, B. J. Am. Chem. Soc.
2013, 135, 542. doi:10.1021/ja310019x
59.Branco, M. C.; Schneider, J. P. Acta Biomater. 2009, 5, 817.
doi:10.1016/j.actbio.2008.09.018
60.Hirano, Y.; Mooney, D. J. Adv. Mater. 2004, 16, 17.
doi:10.1002/adma.200300383
61.Vemula, P. K.; Cruikshank, G. A.; Karp, J. M.; John, G. Biomaterials
2009, 30, 383. doi:10.1016/j.biomaterials.2008.09.045
62.Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer, R. Nat. Nanotechnol. 2007, 2, 751.
doi:10.1038/nnano.2007.387
63.Liang, G.; Yang, Z.; Zhang, R.; Li, L.; Fan, Y.; Kuang, Y.; Gao, Y.;
Wang, T.; Lu, W. W.; Xu, B. Langmuir 2009, 25, 8419.
doi:10.1021/la804271d
64.Ma, M.; Kuang, Y.; Gao, Y.; Zhang, Y.; Gao, P.; Xu, B.
J. Am. Chem. Soc. 2010, 132, 2719. doi:10.1021/ja9088764
917