A smart supramolecular hydrogel of Nα-(4-n- alkyloxybenzoyl)-l-histidine exhibiting pH-modulated properties

Article abstract of DOI:10.1021/la904540x

Six l-histidine-based amphiphiles, N^α-(4-n- alkyloxybenzoyl)-l-histidine of different hydrocarbon chain lengths, were designed, synthesized, and examined for their ability to gelate water. Four of members of this family of amphiphiles were observed to form thermoreversible hydrogels in a wide range of pH at room temperature. The structural variations were characterized by critical gelation concentration, gelation time, gel melting temperature (T_gs), rheology, and electron microscopy. Among the amphiphiles, the n-octyl derivative showed better gelation ability in the studied pH range. The amphiphiles were found to have T_gs higher than body temperature (37 °C) showing their stability. Also, relatively higher yield stress (>1000 Pa) values of the hydrogels show their higher strength. The effective gelator molecules self-assemble into fibrous structures. Scanning electron microscopic picture of the hydrogels revealed large ribbons with right-handed twist. Small-angle XRD and circular dichroism spectroscopy were also employed to characterize the hydrogels. It was observed that π-π stacking, hydrophobic interaction, amide hydrogen bonding, and solubility factor contribute to the stability and strength of the hydrogels.

Full text of DOI:10.1021/la904540x

pubs.acs.org/Langmuir
© 2010 American Chemical Society
A Smart Supramolecular Hydrogel of Nr-(4-n-Alkyloxybenzoyl)-L-histidine
Exhibiting pH-Modulated Properties
Trilochan Patra, Amrita Pal, and Joykrishna Dey*
Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India
Received December 2, 2009. Revised Manuscript Received March 30, 2010
Six L L-histidine of different hydrocarbon chain lengths,
-histidine-based amphiphiles, NR-(4-n-alkyloxybenzoyl)-
were designed, synthesized, and examined for their ability to gelate water. Four of members of this family of amphiphiles
were observed to form thermoreversible hydrogels in a wide range of pH at room temperature. The structural variations
were characterized by critical gelation concentration, gelation time, gel melting temperature (Tgs), rheology, and
electron microscopy. Among the amphiphiles, the n-octyl derivative showed better gelation ability in the studied
pH range. The amphiphiles were found to have Tgs higher than body temperature (37 °C) showing their stability. Also,
relatively higher yield stress (>1000 Pa) values of the hydrogels show their higher strength. The effective gelator
molecules self-assemble into fibrous structures. Scanning electron microscopic picture of the hydrogels revealed large
ribbons with right-handed twist. Small-angle XRD and circular dichroism spectroscopy were also employed to
characterize the hydrogels. It was observed that π-π stacking, hydrophobic interaction, amide hydrogen bonding, and
solubility factor contribute to the stability and strength of the hydrogels.
Introduction
Consequently, during the past 20 years, multistimuli-responsive
hydrogels were investigated.5 But most of these hydrogels are
formed by polymers. To date, a wide variety of hydrogels of
natural or synthetic polymershavebeenreported. Because of their
superior material properties,6 polymeric hydrogels were given
importance in comparison to low-molecular-weight hydro-
gelators (LMWHs). However, constrained synthesis, chemical
cross-linking, thermosetting nature, toxicity, and slow response to
external stimuli7 limitapplicationsof thesehigh-molecular-weight
polymeric gels. The “supramolecular gels” or “physical gels”1a of
LMWHs, on the other hand, offer various advantages over the
conventional polymer gels as gelator molecules are assembled
together by noncovalent forces, such as electrostatic, hydrogen-
bonding (abbreviated H-bonding, hereafter), dipole-dipole,
π-π stacking, and hydrophobic/van der Waals interactions.
For example, one can easily control gel characteristics by chan-
ging pH, temperature, and composition of the aqueous solutions.
Since the cross-links between fibers are noncovalent in nature,
LMWHs exhibit thermoreversibility, rapid response to external
stimuli, and bidegradability. It should be noted that for hydro-
gelation by LMWHs hydrophobic forces are more important.8
Examples of small organic molecules (LMWHs) that sponta-
neously gelate waterare very less and often found by accident. But
such molecules have drawn considerable attention in the past two
decades.9 The subject has been recently reviewed by Weiss and
Terech.9j Among these LMWHs, amino acid-based amphiphiles
have been paid much attention in recent past because of their
biocompatibility and eco-friendly nature.9h-j,10 As a part of the
Hydrogels are a class of soft materials which are prevalent in our
daily life. But they continue to attract considerable atten-
tion of present-day chemists and biologists in terms of their versatile
applications in agricultural, environmental, and medical uses.1 In
particular, hydrogels are of great interest because of their capability
to entrap a large number of water molecules per one gelator mole-
cule and exhibiting swelling, which is suitable for their use in
biomedical applications and tissue engineering,1b,2 biosensing,3a-c
nanotechnology,3d-f controlled drug release,1f,4a-c gene deli-
very,4d-f and water pollution control.4g-i For applications like
drug controlled-release systems or bioseparations, hydrogels are
required to respond to stimuli such as temperature, pH, and ions.
*Author for correspondence: Ph 91-3222-283308; Fax 91-3222-255303;
e-mail joydey@chem.iitkgp.ernet.in.
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Langmuir 2010, 26(11), 7761–7767
Published on Web 04/09/2010
DOI: 10.1021/la904540x 7761

Article
Chart 1. Molecular Structures of Cn-OBH Amphiphiles
Patra et al.
ongoing research carried out in our laboratory in the direction of
self-assembly formation by amino acid-based amphiphiles and
their probable use in drug delivery,9h,9i,11 we have recently shown
that N-(4-n-alkyloxybenzoyl)-
efficient hydrogelators in a wide pH range.12
dipeptide containing β-alanine and -histidine residues. Histidine
L-carnosine amphiphiles act as
L-Carnosine is a
L
is considered to be essential in human infants and becomes a
nonessential amino acid for the adult. The presence of histidine in
protein cause buffering and is a common coordinating ligand in
metalloproteins and is a part of catalytic sites in certain en-
zymes.13 Monohistidylated cationic amphiphiles have been re-
ported to have remarkable gene transfection properties via the
endosome-disrupting characteristics of the histidine functional-
Figure 1. Photographs of vials showing (a) gelation of C8-OBH
at pH 2.0, 8.0, 10.0, and 12.0 in 20 mM phosphate buffer and
(b) pH-reversible gelation.
of histidine-based amphiphiles has been studied earlier,17,19 but
the gelation ability was not fully explored.19b,20 The major
objectives of this study are (i) to examine gelation abilities of
Cn-OBH amphiphiles and (ii) to investigate the effects of envir-
onmental factors such as pH and temperature on the gelation
process. The hydrogels have been characterized by a number of
techniques including electron microscopy, XRD, rheology, and
circular dichroism spectroscopy.
ities.14 On the other hand, comicelles of N-acyl-
L-histidine and
various cationic surfactants have been found to be very effective
in stereoselective micelle-catalyzed deacylation of long-chain
amino acid esters.15 Antioxidant activity toward lipid peroxida-
tion and excellent emulsifying activity of N-acyl-L-histidine have
also been reported.16 In fact, histidine-derived amphiphiles be-
have as an unusual surfactant because the hydrophilic part
contains a carboxylate group and an imidazole side chain,
offering the molecule pH-responsive properties.17 This led us to
design, synthesize, and examine gelation abilities of a series of
Results and Discussion
Gelation Behavior. The gelation of the amphiphiles C6-OBH,
C8-OBH, C10-OBH, C12-OBH, C14-OBH, and C16-OBH was tested
in different pH of 20 mM phosphate buffer (Figure 1). We found
that the C-14 and C-16 analogues remained insoluble in all pH
employed, even when strongly heated at 100 °C. Also, the C-6, C-8,
C-10, and C-12 derivatives remained insoluble at pH 4 and 5 even
when strongly heated in a hot water bath. However, the amphiphiles
C6-OBH, C8-OBH, C10-OBH, and C12-OBH showed gelation in
pH 2 and 7-11.On the other hand, shorter chain length amphi-
philes C6-OBH and C8-OBH also showed gelation in pH 3 and
6 and pH 6, respectively. The gelation occurred just by one or two
alternate heating and cooling cycles. For long chain derivatives
C10-OBH and C12-OBH strong heating for longer time was needed
for solubilization and gelation of the compound. As can be seen in
Figure 1a, the hydrogels are white and opaque, indicating the
presence of large aggregates that have sizes comparable to the
wavelengths of visible light. The amphiphiles become readily soluble
in pH 12.5 buffers at room temperature. Interestingly, we observed
that the sol can switch back to the gel form when the pH is adjusted
to the range 7-11 or 1-2 in the hot condition and allowed to cool at
room temperature (Figure 1b). This indicates that the sol-gel
transition is reversible and is an interesting feature of the systems.
Since gelation depends on solubilization in a solvent, it is
directly affected by the structure of the compound. This class of
compounds, which exist mostly in three different forms cationic,
zwitterionic, and anionic (Figure 2), at different pH shows
variation in their solubilities and hence gelation. The gel forma-
tion of the amphiphiles in water was tested by the “stable to
inversion of the test tube” method. The gels formed in all the pH
solutions were optically opaque. The gelation behavior of the
amphiphiles has been summarized in Table 1. In a given solvent,
NR-(4-n-alkyloxybenzoyl)-
L-histidine amphiphiles (Cn-OBH, see
Chart 1 for structures) in water. The pH-responsive properties
of imidazole ring can make histidine derivative to produce
“intelligent gels” or “smart hydrogels”.18 Aggregation behavior
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7762 DOI: 10.1021/la904540x
Langmuir 2010, 26(11), 7761–7767

Patra et al.
Article
This means less number of gelator molecules in the aggregate
and hence formation of small size aggregates. On the other hand,
in the pH range 6-10, the molecules remain mostly in the
zwitterionic form which means less aqueous solubility and in-
crease of gelator-gelator hydrophobic interaction. These result in
a formation of large aggregates that subsequently upon physical
entanglement produce 3-D network structure. The CGC value
can be used to understand the influence of solution pH on
gelation. It was found that for a particular amphiphile the CGC
value is lowest at pH 8 and highest at pH 11.
It should be noted that at a particular pH the lowest CGC value
was obtained with C8-OBH. This suggests that solubilization,
i.e., hydrophilic-lipophilic balance (HLB), in an amphiphile is
responsible for hydrogelation. In the case of C6-OBH, the smaller
hydrocarbon tail increases the solubility and weakens gelator-
gelator interaction, whereas with C10-OBH and C12-OBH long
chain hindered the solubility at all pH, resulting in high CGC.
However, C8-OBH was found to have optimum HLB for better
gelation in all the pH solutions as indicated by the lower CGC
values. So an optimum solubility of the amphiphiles is required
for gelation to occur. Such HLB is well-known in the mole-
cular assembly of biological membranes. For higher alkyl chain
lengths, the high CGC value may also be explained by the partial
tilt or bending of the long hydrocarbon chain, which weakens
the gelator-gelator directional (e.g., H-bonding) forces, thereby
increasing the CGC value.
Microscopic Observations. As discussed earlier, the gelators
form large self-assembled aggregates in water. To obtain visual
images of the gel aggregates of the amphiphiles at a given pH,
the morphology of the dry hydrogels of C6-OBH, C8-OBH, C10-
OBH, and C12-OBH was investigated by the FE-SEM technique.
The morphology shows fibrous network structure which confirms
gelation by these amphiphiles. In a magnified view (Figure 3) we
found these fibers are made up of bundle of helical ribbons. As the
chirality of the gelators is directly translated into the chirality of
the gel fiber, so the chirality of the microstructures can be clearly
visualized showing the right handed helical ribbons for all the
amphiphiles at both lower and higher pHs. The lengths of the
fibrous bundles are of the order of several micrometers and some
time visible to the naked eye in concentrated solution, and their
widths are ranging from 200 nm to 2 μm. It was found that the
ribbons are either cross-linked or running parallel to each other.
Influence of Chirality. The chirality of a gelator molecule
often plays a significant role in controlling and mediating the self-
assembly of the gelator.21 In fact, many gelator molecules include
chiral centers and are considered to be very important for the
gelation process. The gelling ability of a compound of pure
enantiomer is always found to be better than the racemic form.
The chirality is often translated through formation of chiral
aggregates. Many chiral N-acyl amino acid amphiphiles are
known to form helical aggregates in water.22-24 Recently, Weiss
Figure 2. Proton transfer forms of C8-OBH.
Table 1. Results of Gelation Test at Various pH in 20 mM Phosphate
Buffera
CGC (mg/mL)
pH
2.0
C6-OBH
C8-OBH
C10-OBH
C12-OBH
2.5
(328)
4.5
I
2.0
(347)
I
I
I
2.1
1.9
1.5
(341)
2.2
2.5
4.8
S
2.0
(339)
I
I
I
2.3
(337)
I
I
I
3.0
4.0
5.0
6.0
7.0
8.0
I
5.4
5.2
2.8
(321)
2.9
3.1
5.0
S
I
I
2.1
1.5
(336)
3.1
3.2
8.9
S
2.1
1.9
(333)
3.2
3.5
15.1
S
9.0
10.0
11.0
12.0
a Values refer to the critical gelation concentration (CGC, mg/mL)
necessary for gelation at 25 °C; S: solution, I: insoluble. Values in the
parentheses represent Tgs in K.
gelation occurred when a critical concentration, called critical
gelation concentration (CGC), of the gelator is reached. It is
defined as minimum amount of gelator that can gelate maximum
volume of solvent. The CGC values (mg/mL) necessary for
gelation at various pH for the amphiphiles are shown in Table 1.
It is important to note that the CGC values are less than 10 mg/
mL, which corresponds to gelation capacity of less than 1% (w/v).
In other words, as many as 2000 mol of water molecules is trapped
by 1 mol of gelator molecules. This means that the amphiphiles
are among the most efficient amino acid-based amphiphilic
hydrogelators. At room temperature and at gelator concentration
close to CGC, it took 10-12 h for the gelation to take place.
However, with the increase of the amount and chain length of the
gelator, the gelation kinetics becomes faster. The hydrogels
remained unchanged for more than 2 months when preserved
under constant conditions at low temperature (20 °C). Interest-
ingly, the hydrogels having gelator concentration around the
CGC value shrunk by expelling water after a week or so when
stored at temperature greater than 20 °C.
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Influence of pH and Hydrocarbon Chain Length on Gela-
tion. Since the compound exists in different forms (seeFigure2) at
different pH, the solution pH has a definite role in gelation of
amphiphiles. Since at pH > 12 the gelator molecule exists in the
anionic form, the gelator-gelator interaction is weakened due
to ionic repulsion and thus becomes more soluble in water.
Langmuir 2010, 26(11), 7761–7767
DOI: 10.1021/la904540x 7763

Article
Patra et al.
Figure 3. FE-SEMimagesofthe dry hydrogels ofC6-OBH, atpH2.0 (a), pH8.0 (b), and pH10.0 (c) and ofC8-OBHatpH2.0 (d), pH8.0(e),
and pH 10.0 (f).
and co-workers have studied the kinetics of self-assembled fib-
rillar network formation of p-nitrobenzyl arjunolate in mixed
organic solvent by CD spectroscopy.25 The chirality-induced
helicity in molecular self-assembly of C8-OBH is clearly observed
in the micrograph b of Figure 3. Homochiral interactions between
histidine headgroups of the gelators perhaps facilitate one-
dimensional (1-D) growth of the fibrous aggregates, the physical
entanglement of which leads to the 3-D network structures. The
homochiral interaction among chiral amphiphiles should lead to
the formation of 1-D helical fibers, which can be further con-
firmed by the characteristic circular dichroism (CD) spectrum.26
Figure 4. Circular dichroism spectra of 2.0 mM C8-OBH in
phosphate buffers of pH 8 and 12 and in methanol.
Since the hydrogel formed by C8-OBH is optically opaque, the
aqueous solution of the amphiphile was analyzed for chiral
organization by CD spectroscopy. Figure 4 shows the CD spectra
shown in Figure 3. These peaks are due to the length of a repeat
unit along the long axis of the molecule. The XRD data suggest
that in all the three pH the hydrogels have similar morphology
and are crystalline in nature.
of C8-OBH in methanol and in pH 8 and 12 having concentration
much less than CGC. The negative band in the wavelength range
215-240 nm of the CD spectrum in pH 8 solution supports the
existence of helical aggregates. The formation of helical aggre-
gates was also observed with the structurally similar amphiphile,
Viscoselastic Behavior. During the gelation test, it was
observed that the hydrogels in all the pH-containing gelators
corresponding to the CGC value partially broke down and part of
the trapped water was expelled out, even upon gentle shaking of
the vial. This indicated that the hydrogels are less rigid. The
rheological properties of the hydrogels of C8-OBH as a repre-
sentative example were studied at three different pH values. The
mechanical strength of a gel which is measured by the storage
modulus (G0) and loss modulus (G00) is dependent on gelator
concentration. We measured these quantities as a function of
applied stress (σ) and frequency on the hydrogels of C8-OBH in
pH 2, 8, and 10 that contained gelator of ca. 5 times its CGC
value. Figure 6 shows the variation of G0 and G00 with strain
frequency. It is seen that in all the pH both G0 and G00 are almost
independent of frequency which is characteristic of gel struc-
ture. Also at any given frequency, the G0 much higher than G00,
sodium N-[4-n-dodecyloxybenzoyl]-L
-valinate.27 Since even dilute
solution of C8-OBH is not transparent, we could not measure the
CD spectrum in pH 2.0 solution.
X-ray Diffraction Studies. The XRD patterns of the
air-dried gel cast film of C8-OBH in pH 2.0, 8, and 10.0 as
representative example have been depicted in Figure 5. From the
peak positions (2θ values) corresponding planes and interplanar
distances (d) were calculated using Bragg’s equation. The hydro-
gel of C8-OBH exhibits periodical diffraction peaks with their
positions approximately at a ratio of 1:2:3:4, which suggests an
ordered lamellar phase. This isconsistent with the FESEM images
(25) Bag, B. G.; Dinda, S. K.; Dey, P. P.; Mallia, A.; Weiss, R. G. Langmuir
2009, 22, 8663.
(26) Helfrich, W.; Prost, J. Phys. Rev. A 1988, 38, 3065.
(27) Mohanty, A.; Dey, J. Langmuir 2004, 20, 8452.
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Article
Figure 6. Variation of storage modulus (G0) and loss modulus
(G00) with frequency (f) of hydrogel C8-OBH in pH 2.0, 8.0, and
10.0 at 298 K.
Figure 7. Variation of storage modulus (G0) and loss modulus
(G00) with shear stress (σ) of hydrogel C8-OBH in pH 2.0, 8.0, and
10.0 at 298 K.
Figure 5. X-ray diffraction pattern of hydrogel film of C8-OBH at
(a) pH 2, (b) pH 8, and (c) pH 10.
thus disfavors gel formation. We have determined the gel melting
temperature that is gel-to-sol transition temperature (Tgs) of the
hydrogels (Table 1) at different pH, keeping the amount of gelator
(15 mM) fixed. The gel melting temperature was determined by
placing the screw-cap glass vials containing gels in a temperature-
controlled water bath and visually observing the flow upon tilt for
every degree rise in temperature. Although the gel structure was
found to melt upon heating, the gelationof the solutiontookplace
upon cooling to room temperature. That is, the gels are thermo-
reversible. All the hydrogels have Tgs above 37 °C. Thus, the
hydrogels formed by different amphiphiles at various pH were
found to be thermally quite stable. The variation of Tgs with the
number of carbon atoms (NC) of the hydrocarbon chain of
the gelators has been shown by the bar graph in Figure 8a. It
has been found that for any gelator Tgs is less in pH 2 than in pH 8.
This is consistent with the yield stress values. Also, at any given
pH, Tgs was found to be highest for C8-OBH. The gel melting
temperature of C8-OBH was also measured at various concentra-
tions in pH 8 as well as in pH 2. Figure 8b shows the plots of
Tgs versus [C8-OBH]. It is observed that the Tgs value increases
linearly with [C8-OBH] in the concentration range studied.
The Tgs value is consistent with the lower CGC value of C8-
OBH. For C10-OBH and C12-OBH, perhaps, lower solubility or
partial folding of the hydrocarbon chain or both hinder growth of
self-assemblies. Thismeans formationofshort fibers whichresults
in less entanglement of the fibers and hence higher CGC value and
lower yield stress and Tgs values. The increase of concentration,
on the other hand, facilitates growth of the fibers, thereby causing
more entanglement. This is reflected by the increase of Tgs value.
indicating more elastic nature of the gels like solids. The difference
between G0 and G00 can be taken as an indicator of the rigidity or
stability of a gel. The (G0 - G00) values were ca. 98220, 342 308,
and 61921 Pa for the hydrogels in pH 2, 8, and 10, respectively.
This means that the hydrogel in pH 8 is more firm and rigid than
hydrogel in pH 2 which in turn more firm than the gel in pH 10.
Figure 7 shows the plots of G0 and G00 versus applied stress (σ) at a
constant frequency of 1 Hz. It can be observed that above a
critical stress value both G0 and G00 abruptly fall to a very low
value, indicating flow of the hydrogel. This critical stress value is
referred to as yield stress (σy). The σy values at pH 2, 8, and 10 as
obtained from the breakpoint of the respective plot are respec-
tively, 2662, 4830, and 1190 Pa. The σy values are quite large,
suggesting high mechanical strength of the hydrogels. However,
the hydrogel in pH 8 has the highest yield stress value that
decreases both upon decrease and increase of the acidity of water.
Such modulation in viscoelastic properties with pH is an inter-
esting feature of the system and has significant value as far as drug
release is concerned. The change in viscoelastic properties can be
attributed to the variation of supramolecular interactions and
hence 3-D network structure in the hydrogel at different pH.
Thermal Stability of the Hydrogels. It is well established
that gel formation is strongly dependent on the gelator concen-
tration and temperature. Since higher gelator concentration
promotes growth self-assemblies, gelation is favored. On the
other hand, higher temperature dismantles self-assemblies and
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DOI: 10.1021/la904540x 7765

Article
Patra et al.
Figure 8. (a) Variation of gel melting temperature (Tgs) with
hydrocarbon chain length (NC) at pH 2 and pH 8. (b) Plot of gel
melting temperature (Tgs) versus [C8-OBH] at pH 2 (2) and
pH 8 (9).
Figure 9. Temperature-dependent chemical shift (δH in ppm) of
imidazole (Imz) proton (9) and phenyl proton (2) in 10 mM
C8-OBH gel in 20 mM phosphate buffer of pH 8.
Driving Force for Aggregate Formation. It is well-known
that lipophilic (hydrophobic) interactions between gelator mole-
cules in strongly H-bonding and polar solvents provide a major
contribution to the overall stabilization of the assemblies while
H-bonding usually gives a dominant contribution in lipophilic
solvents.28 The role of amide H-bonding in the self-assembly
formation in water by N-acyl amino acid amphiphiles has
already been reported in the literature.23,24 In fact, we have
already demonstrated intermolecular H-bonding in the formation
of bilayer self-assemblies by a structurally similar amphiphile,
sodium N-[4-n-dodecyloxybenzoyl]-L
-valinate.27 Further, to
examine the possible role of lipophilic interactions, including
π-π stacking in aggregate formation by C8-OBH, we have
1
measured variable temperature (298-343 K) H NMR spectra
1
Figure 10. Time-dependent release (f) of NS (9) and TCH (2) in
20 mM phosphate buffer (pH 6) from 8 mM C8-OBH gel in 20 mM
phosphate buffer of pH 8, containing 5 ꢀ 10-5 M drug each.
of its hydrogel prepared in D2O solvent. The representative H
NMR spectra of C8-OBH (ca. 0.5% w/v, in D2O) at temperatures
298 and 343 K have been shown in Figure S1 of the Supporting
Information. Usually the signals of gelator molecules assembled
in the 3-D gel network are not observed due to long correlation
times. Thus, the observed gelator signals are due to the smaller
assemblies dissolved in the entrapped D2O solvent. The π-π
stacking interaction can be confirmed from the change in chemi-
cal shift (δ) of the aromatic protons. The signals of the phenyl
protons (PhH) of C8-OBH molecule have merged into one broad
band even at elevated temperatures, confirming the assembled
state. The signals of imidazole ring protons (ImzH), however, are
well separated. The δH values of both types of protons are
observed to shift toward downfield positions linearly (Figure 9)
with the increase of temperature. The increase ofδH value with the
increase of temperature suggests a change in the microenviron-
ment of amphiphiles due to loss of H-bonding and π-π stacking
interactions at higher temperatures. The necessity of π-π stack-
ing is also confirmed by the failure of hydrogel formation by
to demonstrate the potential application of the hydrogel of
C8-OBH in drug delivery, we describe the entrapment and release
of two small drug molecules: naproxen sodium salt (NS) and
tetracycline hydrochloride (TCH). NS is an anti-inflammatory
drug, while TCH has antimicrobial activity. TCH was chosen
since it can interact with the gelator not only via van der Waals
interactions and H-bonding but also more strongly with the
carboxylate group of the gelator since it possesses an ammonium
group. Consequently, it may be retained within the hydrogel to a
greater extent than NS which can interact with the gelator only via
weaker van der Waals interactions. Further, both molecules are
fairly soluble in water (pH 8) and UV-active, making their release
from the hydrogel easy to follow by spectrophotometry.
Thus, 8 mM of C8-OBH in 1.0 mL of phosphate buffer
containing 5 ꢀ 10-5 M NS (or TCH) was dispersed in an
ultrasonic bath for 5 min followed by three heating-cooling
cycles. The mixture was then transferred into a quartz cuvette
(1 cm2) and left at room temperature for gelation to occur.
Although the homogeneous mixture turned into gel within a
few minutes, it was allowed to stand for equilibration for 1 h at
room temperature, after which 2 mL of 20 mM phosphate buffer
(pH 6.0) was carefully added on top of the hydrogel with the help
of a micropipet. Absorbance of the solution on top of the gel
phase was measured at 330 nm (or 360 nm for TCH) at different
time intervals. The amount released at any time was calculated as
fraction of the total amount dissolved in the gel phase. Assuming
that the release of the drug from the gel over time follows first-
order kinetics, the data sets were fitted to eq 1:
the structurally similar amphiphile, NR-dodecanoyl-
L-histidine
(L-C12His), which does not have a phenyl group in the hydro-
carbon chain. Thus, within the bilayer leaflet, the amphiphiles are
connected by intermolecular H-bonds to form H-bond network
to develop the superstructures in which the aromatic rings are
stacked on top of each other. The detailed microstructural
characterization of the hydrogels by spectroscopic and micro-
scopic studies as described above shows that a precise balance
between hydrophilicity and hydrophobicity is the key factor
in this self-assembling process, which involves intermolecular
H-bonding and hydrophobic interactions.
Drug Entrapment and Release Studies. The water-
immobilizing ability and the hydrophobic domains in the bilayer
aggregates make these hydrogels potential drug carriers. In order
f ¼ 1 - e- kt
ð1Þ
where f is the fraction of NS (or TCH) at time t in the solution and
k is the first-order rate constant. A reasonably good fit can be
(28) Zweep, N.; Hopkinson, A.; Meetsma, A.; Browne, W. R.; Feringa, B. L.;
van Esch, J. H. Langmuir 2009, 25, 8802.
7766 DOI: 10.1021/la904540x
Langmuir 2010, 26(11), 7761–7767

Patra et al.
Article
Synthesis of Amphiphiles. NR-[4-n-Alkyloxybenzoyl]-
-his-
observed in Figure 10 for both the drug molecules. The values of k
L
for NS and TCH were found to be 4.2 ꢀ 10-4 and 7.5 ꢀ 10-5 s-1
,
tidine amphiphiles (C8-OBH, C10-OBH, and C12-OBH) were
synthesized following a procedure described elsewhere.29 The
compounds were purified by recrystallization from acetone-
water or ethanol-water mixture. For NR-[4-n-hexyloxybenzoyl]-
respectively. Thus, the release rate of the entrapped material from
the hydrogel of C8-OBH is observed to be relatively slow for both
drug molecules. However, as expected, the release rate of TCH is
10 times slower than that of NS. It is observed that at pH 6 only
about 50% and 10% of the entrapped NS and TCH, respectively,
were observed to be released within 6 h. It should be noted that no
change in the gel phase occurred during the release process.
Therefore, it can be concluded that the release of entrapped drug
was entirely due to diffusion.
L
-histidine (C6-OBH), however, L-histidine was directly reacted
with hexyloxybenzoyl chloride in the THF/H2O mixture in the
presence of triethylamine (pH 8-9). Chemical identification of all
the compounds was performed by use of H NMR and FT-IR
1
spectroscopy. The details of chemical identification are available in
the Supporting Information.
Methods and Instrumentation. The melting point of solid
compounds was measured using the Instind (Kolkata) melting
point apparatus with open capillaries. The measurements of
optical rotations were performed with a JASCO (model P-1020)
digital polarimeter. The FT-IR spectra were measured with a
Perkin-Elmer(model Spectrum Rx I) spectrometer. The 1H NMR
spectra were recorded on an AVANCE DAX-400 (Bruker,
Sweden) 400 MHz NMR spectrometerinCD3OD orD2O/NaOD
solvent with CH3CN as a standard. The circular dichroism (CD)
spectra were measured with a JASCO J-810 spectropolarimeter
using a quartz cell with a path length of 1.0 mm. The kinetics of
drugrelease was studied byuse of a Shimadzu(UV-2450) UV-vis
spectrophotometer. All measurements were done at 298 K unless
otherwise mentioned.
Gelation was studied by dissolving 5 mg of solid gelator in a
screw-cap vial in requisite volume of 20 mM phosphate buffer by
heating in a hot water bath (∼70 °C) and subsequently allowed to
cool at 25 °C in a temperature-controlled water bath. Resistance
of the mixture to flow under gravity on inversion of the vial
indicated gel formation. Melting temperature of the hydrogels
was determined by inverted-tube experiment in which the screw-
cap vial containing the gel was put in a temperature-controlled
water bath (JULABO, model F12). The temperature of the bath
was gradually increased at a rate of 1 deg/min, and the tempera-
ture was noted where the gelated mass started to flow on tilting of
the vial. Each experiment was repeated at least twice. The melting
temperature did not vary more than (1 °C.
For electron micrographs, the hot sample solution was placed
on the aluminum or copper foil, allowed to cool, and air-dried at
room temperature. The gel cast films were further dried in
desiccators for 24 h. A layer of gold was sputtered on top to make
conducting surface, and finally the specimen was transferred into
the field emission scanning electron microscope (FESEM, Zeiss,
Supra-40) operating at 5-10 kV to get the micrograph.
The XRD spectra were taken at room temperature for all air-
dried hydrogel samples prepared on a glass slide. The experiment
was performed on a Pan analytica X’ Pert pro X-ray diffract-
ometer using Cu target (Cu KR) and Ni filter at a scanning rate of
0.001 s-1 between 2° and 12°, operating at a voltage of 40 kV and
current 30 mA.
For rheology measurements were performed on a Bohlin RS
D-100 (Malvern, UK) rheometer using parallel-plate (PP-20)
geometry. The gap between the cone and plate was fixed at
300 μm. The hydrogel was placed on the rheometer, and a stress-
amplitude sweep experiment was performed at a constant oscilla-
tion frequency of 1.0 Hz at 25 °C.
Conclusions
In summary, a family of low-molecular-weight L-histidine-
derived hydrogelators (Cn-OBH) that exhibit efficient gelation
capability have been synthesized. Among these C8-OBH acts as
an efficient gelator in pH 8.0; the hydrogel of C8-OBH was found
to have the highest thermal and mechanical stability. The pH-
dependent reversibly modulated gelation properties of the ther-
moreversible hydrogels have been reported. Indeed, reversible
switching between gel and sol (or precipitate) could be easily
controlled by changing pH. The pH dependence of CGC and gel
melting temperature Tgs suggests that gelation is driven by
hydrophobic, hydrogen-bonding, and π-π stacking interactions.
The presence of protons changes supramolecular interactions by
forming and breaking intermolecular hydrogen bonds, which
controls the degree of branching and interfacial tension in
the 3-D hydrogel network. The gelator molecules self-assemble
to form 1-D bilayer structures (ribbons) of high aspect ratio in all
pH. The chirality of the gelator molecules is translated through
formation of chiral aggregates, such as twisted ribbons as man-
ifested by the characteristic CD spectrum and SEM pictures. The
ability to form hydrogels by NR-(4-n-alkyloxybenzoyl)-
amphiphiles has demonstrated that for the pH-dependent hydro-
gel formation by N-(4-n-alkyloxybenzoyl)- -carnosine amphi-
philes the β-alanine residue of -carnosine has no significant
role. The hydrogels of the -histidine amphiphiles have high yield
stress showing good mechanical strength and exhibit pH-respon-
sive viscoelastic properties. The present study generated a new
type of hydrogel and showed a facile route for modulat-
ing mechanical properties of supramolecular hydrogels. The gel
melting temperature, Tgs (concentration-dependent), was ob-
served to be much higher than body temperature (37 °C). Slow
release ofthe anti-inflammatorydrugnaproxenand antimicrobial
drug tetracycline shows that the hydrogel may find applications in
transdermal drug delivery. Further work in this direction is
currently underway in this laboratory.
L-histidine
L
L
L
Experimental Section
Materials. 4-Hydroxybenzoic acid, 1-bromododecane, 1-bromo-
decane, 1-bromooctane, anhydrous potassium carbonate, sodium
bicarbonate, N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbo-
diimide (DCC), L-histidine, sodium hydrogen carbonate, sodium
Acknowledgment. Financial support (Grant SR/S1/PC-18/
2005) from Department of Science and Technology, New Delhi,
is duly acknowledged. T.P. thanks CSIR, India, for a research
fellowship. We thank Dr. Dhara of the School of Medical Science
and Technology, IIT, Kharagpur, for assistance with the rheo-
logical measurements.
dihydrogen phosphate, disodium hydrogen phosphate, and
sodium hydroxide were purchased from SRL, Mumbai, India,
and were used without further purification. Hexyloxybenzoyl
chloride was obtained from Aldrich. All the organic solvents were
of highest purity commercially available and were dried and
distilled fresh before use. Milli-Q water was used for the prepara-
tion of buffers. All the surfactants employed in this study were
synthesized in the laboratory as described below.
Supporting Information Available: Details of chemical
identification of the amphiphiles including spectral data and
1H NMR spectra at different temperatures. This material is
available free of charge via Internet at http://pubs.acs.org.
(29) (a) Mohanty, A.; Dey, J. J. Chem. Soc., Chem. Commun. 2003, 1348.
(b) Mohanty, A.; Dey, J. Langmuir 2007, 23, 1033.
Langmuir 2010, 26(11), 7761–7767
DOI: 10.1021/la904540x 7767