Single amino acid based thixotropic hydrogel formation and pH-dependent morphological change of gel nanofibers

Article abstract of DOI:10.1039/c3sm27050e

A single amino acid (phenylalanine) based pyrene conjugated low molecular weight hydrogelator has been discovered. This amino acid derivative has been found to form hydrogels in a wide range of aqueous solutions of pH 7.46-14 and the estimated minimum gelation concentration (MGC) in phosphate buffer solution at physiological pH 7.46 is 0.037% (w/v) i.e. 0.85 mM. A transparent hydrogel has been produced in the pH range (7.46-10.5), while a translucent hydrogel has been formed at higher pH. The hydrogel has been characterized by field emission scanning electron microscopy (FE-SEM), fluorescence microscopy, differential scanning calorimetry (DSC), circular dichroism (CD), X-ray diffraction (XRD) and rheological studies. A distinct morphological change of the gel nanofibers from helical to tape-like morphology has been noticed in the FE-SEM studies with an increase in pH. These observations have been supported by a significant decrease of the CD signals and different XRD patterns found with the increase in pH. Interestingly, this hydrogel shows a pH dependent thixotropic property. The thixotropic property of the hydrogel has been examined using rheological experiments. This thixotropic property of the hydrogel has been utilized for the encapsulation of vitamin B₁₂ and an anticancer drug, doxorubicin, in the hydrogel matrix without any heating-cooling cycle and sustained release of these bioactive molecules has been studied at physiological temperature (37 °C) and pH (7.46) over a period of about 3 days.

Full text of DOI:10.1039/c3sm27050e

Soft Matter
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Single amino acid based thixotropic hydrogel formation
and pH-dependent morphological change of gel
nanofibers†
Cite this: DOI: 10.1039/c3sm27050e
Jayanta Nanda, Abhijit Biswas and Arindam Banerjee*
A single amino acid (phenylalanine) based pyrene conjugated low molecular weight hydrogelator has
been discovered. This amino acid derivative has been found to form hydrogels in a wide range of
aqueous solutions of pH 7.46–14 and the estimated minimum gelation concentration (MGC) in
phosphate buffer solution at physiological pH 7.46 is 0.037% (w/v) i.e. 0.85 mM. A transparent hydrogel
has been produced in the pH range (7.46–10.5), while a translucent hydrogel has been formed at
higher pH. The hydrogel has been characterized by field emission scanning electron microscopy (FE-
SEM), fluorescence microscopy, differential scanning calorimetry (DSC), circular dichroism (CD), X-ray
diffraction (XRD) and rheological studies. A distinct morphological change of the gel nanofibers from
helical to tape-like morphology has been noticed in the FE-SEM studies with an increase in pH. These
observations have been supported by a significant decrease of the CD signals and different XRD
patterns found with the increase in pH. Interestingly, this hydrogel shows a pH dependent thixotropic
property. The thixotropic property of the hydrogel has been examined using rheological experiments.
This thixotropic property of the hydrogel has been utilized for the encapsulation of vitamin B12 and an
anticancer drug, doxorubicin, in the hydrogel matrix without any heating–cooling cycle and sustained
Received 4th September 2012
Accepted 5th February 2013
DOI: 10.1039/c3sm27050e
ꢀ
release of these bioactive molecules has been studied at physiological temperature (37 C) and pH
www.rsc.org/softmatter
(7.46) over a period of about 3 days.
molecular building blocks. Information regarding the self-
assembly is encoded by the amino acid sequence. Moreover, the
Introduction
Amino acid and short peptide derivatives have attracted
considerable research interest due to their facile synthesis,
variation in amino acid sequence and easy functionalization.
structural and functional properties of short peptide based
hydrogels can be modulated by changing the peptide sequen-
1
6c
ce. There are several reports on hydrogels formed by dipep-
These molecules have unique properties to self-assemble into tides and tripeptides with different N-terminally conjugated
3i,6
7
8a
different structures, mimicking natural architectures such as b- aromatic groups including Fmoc, napthyl, Cbz and pyr-
2
8b–e
sheets and a-helices in solution and also to form gels under enyl.
However, single amino acid containing hydrogels are
3,4
9,10
suitable conditions. Supramolecular gels are an important relatively much less reported in literature.
Xu, Adams and
class of so materials consisting of a lot of entrapped solvent
others have reported amino acid based supramolecular hydro-
gels. Nilsson and his group have studied the self-assembly and
gelation of Fmoc conjugated ring substituted phenylalanine
9
molecules within the brous network structure formed by the
3,4
self-assembly of gelator molecules. Over the years, amino
acid, peptide and other naturally occurring building block derivatives.10b–e Phenylalanine residue is an excellent moiety to
10a
10b–e,11
based gels are gaining increasing interest due to their study self-assembly and gelation.
The side chain of the
5
biocompatibility and biodegradability. Short peptide based phenylalanine (Phe) residue is aromatic as well as hydrophobic
hydrogels have attracted immense interest in so-biomaterial in nature. It plays a vital role in self-assembly and gelation as
research. Amino acids and peptide molecules are smart the introduction of this residue promotes both p–p stacking
interactions and hydrophobic interactions in self-assembly and
10
the hydrogelation process. Gelator molecules are self-associ-
Department of Biological Chemistry, Indian Association for the Cultivation of Science,
Jadavpur, Kolkatta-700032, India. E-mail: bcab@iacs.res.in; arindamkol1966@gmail. ated to form different nanostructures like tape, rod, helical
com; Fax: +91-33-2473-2805
3,4
nanobers, twisted nanobers and others. All these nano-
†
Electronic supplementary information (ESI) available: NMR and HRMS spectra
structures are formed through co-operative effects of various
weak non-covalent interactions and these non-covalent inter-
actions are stimuli responsive. The morphological change of
these nano-architectures of self-assembled materials can be
of the pyrene conjugated amino acid derivatives, recovery of gel strength at
different pHs, SEM image of recovered gel materials, FL microscopic image at
wet gel, FT-IR of solid gelator and wet gel, and drug encapsulation study within
gel matrix. See DOI: 10.1039/c3sm27050e
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tuned by different stimuli including the solvent, temperature, in nature and shows a thixotropic property. For the super-
2
b,f,12–14
pH, light, or doping of foreign materials.
the morphology of an azo group containing an amphiphilic exceptionally low, below 0.1% (w/v).
hydrogel has been observed from double helical nanobers to knowledge, there is no report of a single amino acid based
Modulation of gelator, the minimum gelation concentration of the gelator is
17b
To the best of our
3f,o,17
spherical micelles, vesicles or unfolded nanobers by external pyrene conjugated hydrogel with a super-gelation efficiency
12a
stimuli, such as pH, light, and surfactant addition. Similarly, and thixotropic property. Interestingly, this hydrogel material
the morphology of a dipeptide based azobenzene bearing an shows a pH dependent thixotropic property and morphological
amphiphilic hydrogel has been modulated by the salt concen- transformation. The thixotropic property has been studied by
tration and UV light. A recent report from our group has also monitoring the recovery property of the hydrogel with time in a
demonstrated the morphological transformation of synthetic rheometer. The hydrogel has been characterized by eld emis-
tripeptide based nanotubes to vesicles by tuning the pH in sion scanning electron microscopy (FE-SEM) at different pHs
solution and rapture of the nanovesicle upon the deprotonation and this indicates a morphological transformation from helical
of the tyrosine residue at higher pH. Calcium ions, having a nanober to straight nanober (tape-like) upon the increase in
high binding affinity towards the carboxylic acid, transform the pH. A pH-dependent circular dichroism (CD) study shows the
helical morphology of the two component based hydrogel diminishing of CD signals with an increase in pH. The pH-
12b
13a
13b
nanobers into straight nanobers. Stupp and Pashuck have responsive morphological transformation as well as the thixo-
reported the time-dependent morphological transformation of tropic property suggest that the nanoscale morphology and the
12e
peptide amphiphiles from twisted ribbons to helical ribbons.
bulk property of the gel material are dependent on the pH. The
However, in that study, no external stimulus has been used and thixotropic behavior of the hydrogel at physiological conditions
metastable twisted ribbon structures have been transformed allows the encapsulation of bioactive molecules within the
into thermodynamically stable helical ribbon structures with hydrogel matrix without a heating–cooling process. Then, their
aging. There are several reports on the morphological trans- sustained release in physiological pH and temperature from the
formation of the amino acid, peptide and other molecule based gel matrix have been studied. This indicates a promising use of
12a–d,f,h–k,14
gels.
However, in most of the supramolecular gel these materials as a drug delivery vehicle.
systems, alteration of the stimulus causes a change in the
nanoscale morphology that is associated with a distinct phase
Results and discussion
Gelation and thermal studies
12b,c,f
change from gel to sol state or vice versa.
So, a morpho-
13b,14
logical change in the gel without a phase change is very rare.
In general, gels formed by the LMWGs are broken by
mechanical stress/agitation and they are converted to solution
phase (sol). They are unable to reform the gel material until the
sol is heated to provide a clear homogeneous solution and is
allowed to cool for some time. However, in very few cases,
The gelation property of the pyrene conjugated single amino
acid (Phe) derivative (Scheme 1) has been studied in aqueous
solutions at different pHs. It has been observed that the gelator
molecule could gelify aqueous solutions (Fig. 1) over a wide
range of pHs (7.46–14). The Phe residue was replaced by another
hydrophobic amino acid residue, valine (Val), to address the
question whether this new molecule with the Val residue (Pyr 2)
can be self-assembled to form a gel or not. However, it has been
found that Pyr 2 is unable to form a hydrogel under similar
conditions to that of Pyr 1. This may be due to the presence of
an aromatic Phe residue in Pyr 1, which promotes more p–p
interactions to facilitate self-association and the gelation
15,16
thixotropic supramolecular gels
have been obtained, where
reformation of the gel from the sol has been observed aer
removing the external stress only. Thixotropy is an important
function in the human body. Functions of protoplasm, red
blood cells, synovial uid, and muscular activities are regulated
by thixotropy. Recently, supramolecular hydrogels with a
thixotropic property are getting much attention due to the
16a,b,e,g,h
different biomedical applications.
Yang and coworkers
10b–e,11
process.
have recently reported napthyl acetic acid conjugated peptide
Hydrogels have been prepared by heating the gelator
compound in aqueous solutions of different pHs in glass vials
until it (gelator compound) has been dissolved completely.
Upon cooling at room temperature for about a minute, the total
volume of these homogeneous aqueous solutions of the gelator
have been immobilized to form self-supporting hydrogels
based thixotropic hydrogels and their different biomedical
16a,b
applications.
Ying and coworkers have also studied three
dimensional cell cultures in a polyethylene glycol–silica (PEG–
silica) based thixotropic hydrogel matrix and the effect of matrix
16g,h
stiffness on stem cell differentiation.
erty allows formulations of the hydrogel to be injected using a
ne needle in a unique injectable hydrogel based drug delivery
The thixotropic prop-

system. Various bioactive agents (drugs, proteins, vaccines or
plasmid DNAs) can be encapsulated inside these hydrogels at
room temperature, which can then be loaded in a syringe and
stored. Owing to the thixotropic nature of these hydrogels, the
drug-loaded formulation can then be injected into the tissue
under pressure.
In this study, we have synthesized a pyrene conjugated
phenylalanine (Phe) derivative (Pyr 1), which is a superhydrogel Scheme 1 Chemical structures of the amino acid based pyrene derivatives.
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Fig. 3 Demonstration of the pH dependent thixotropic property of hydrogels
prepared by using (i) 50 mM phosphate buffer solution at pH 7.46 and (ii)
aqueous solution at pH 14. (a) Hydrogels prepared by the heating–cooling
method (b) the transformation of gel to sol after mechanical shaking; (c) refor-
mation of the hydrogel after keeping the sol for several minutes at pH 7.46, while
no reformation of the hydrogel has been found at pH 14 even after standing the
sol for one day.
Fig. 1 Camera view of the hydrogel obtained from Pyr 1 in 50 mM phosphate
buffer solution at pH 7.46 in a glass vial (a) under normal light and, (b) under UV
light at 365 nm. The concentration of the gelator in the hydrogel is 0.05% (w/v).
(Fig. 1). The minimum gelation concentration (MGC) of this
gelator in phosphate buffer solution at pH 7.46 has been
liquid (sol) has been spontaneously transformed into the gel by
keeping this vial without any shaking or disturbance for >5
minutes, indicating the thixotropic nature of the gel. The same
experiment was repeated several times using that particular gel
vial and further gel formation was observed upon resting. The
same experiment was performed by taking hydrogels prepared
at pH > 10.5. However, no such observation (sol to gel) was
noticed this time (Fig. 3). So, at higher pH (pH > 10.5), these gel
materials did not show any thixotropic property. Therefore, the
thixotropic property of the hydrogel is pH dependent. An
increase of the mechanical shaking time delays further gel
formation of these hydrogels that have been prepared in the
lower pH range. But, it has no effect for these hydrogels that
have been prepared at higher pH (pH > 10.5).
measured to be 0.037% (w/v). This is an example of super-
17
gelator. The thermal stability of the hydrogel prepared using a
phosphate buffer of pH 7.46 has been measured by determining
the gel melting temperature (T ), i.e. the temperature at which
m
the gel is transformed into the solution (sol). The Tm values have
1
8
been checked according to the ‘test-tube inversion’ method
ꢀ
under a heating rate 1 C per 5 minute. Fig. S13 in the ESI†
m
represents the T vs. gelator concentration plot.
Moreover, this hydrogel is thermo-reversible in nature. The
gel has been transformed into solution (sol) upon heating and
again it returns to the gel upon cooling at room temperature for
a few minutes. A differential scanning calorimetry (DSC)
experiment has also been carried out to scrutinize the thermo-
reversibility of the gel (0.5% w/v) at pH 7.46. From the DSC
micrograph (Fig. 2), it has been observed that the gel shows an
exotherm on heating and an endotherm on cooling. The
Rheological study
ꢀ
hydrogel melted at 66.4 C (exothermic peak) and reformed at
To study the visco-elasticity of this gel material, the hydrogel
was subjected to different rheological studies. A rheological
study of the hydrogel is important for determining its utility in
biomedical applications. In this paper, mechanical strength of
these hydrogels has been measured at different pHs (Fig. 4).
Rheological experiments have been performed by using the
hydrogel (0.42%, w/v) prepared in different pH solutions. The
ꢀ
4
2.7 C (endothermic peak). The temperature difference
ꢀ
between the exotherm and the endotherm is 23.7 C, indicating
hysterisis (Fig. 2). This is characteristic of a rst-order phase
18
transition.
The physical looks of these hydrogels are different in
different pH values. In the pH range 7.46–10.5, the hydrogel
formed in phosphate buffer solution is transparent; while in
higher basic pH (pH > 10.5), it forms a translucent hydrogel
0
00
storage modulus (G ) and loss modulus (G ) have been
(Fig. 3). Interestingly, the hydrogel obtained at pH 7.46 (using
phosphate buffer) has shown a thixotropic property. The gel
phase has been transformed into a viscous solution (sol phase)
by mechanical shaking or vortexing. However, this viscous
Fig. 2 DSC thermogram of the hydrogel at 0.5% (w/v) in 50 mM phosphate
buffer solution at pH 7.46.
Fig. 4 Angular frequency sweep experiments of hydrogels prepared at different
pHs.
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measured in frequency sweep experiments. For a gel phase
Similar experimental cycles have been applied for the
0
00
0
00
material, G is greater than G and G ¼ G when the gel starts hydrogel at pH 10.5 (Fig. S17, ESI†). However, the gel strength is
0
0
to break into the sol state. In the sol state, the G is greater not fully recovered at this pH. Up to one cycle, signicant
0
than G . It has been noticed from Fig. 4 that the storage recovery of the gel strength has been noticed. Aer that, the gel
0
00
modulus value (G ) is higher than the loss modulus (G ) in the is not properly recovered from the sol state even aer the
ꢁ1
experimental frequency region (10–100 rad s ) for all hydrogel complete removal of applied stress. In this case, random storage
materials. This suggests the formation of typical ‘so solid- and loss moduli values are observed. The hydrogel prepared at
like’ gel phase materials.
the storage modulus (G ) is increased with an increase of the these rheological experiments lead to the conclusion that gel
pH of the aqueous solution. The storage modulus value of the strength (storage modulus) is increased and the thixotropic
hydrogel prepared at pH 14 has been found at a maximum; property or the gel recovery property from the sol is systemati-
however, it is the least for the hydrogel prepared at 7.46 cally diminished with an increase in pH of the aqueous solu-
18,19
An interesting observation is that pH 14.0 exhibits no recovery property (Fig. S18, ESI†). So, all
0
(Fig. 4).
tion. Interestingly, a good thixotropic property of the gel at
Interestingly, these hydrogel materials exhibit a thixotropic lower pH (pH 7.46) can be utilized for the encapsulation and
property within the pH range 7.46–10.5. Upon mechanical sustained release of biomolecules.
shaking, the gel material is transformed into solution (sol) and
it is reformed into the self-supporting gel state upon
pH dependent morphological study
15,16
resting.
To verify our observation more accurately, rheo-
It is remarkable to notice that these hydrogels prepared at
different pHs look different, as a transparent hydrogel is
observed at pH 7.46 and a translucent hydrogel is formed at
pH 14. This has prompted us to investigate their morphologies
at different pHs. To obtain a better insight into the morphology
of hydrogels, eld emission scanning electron microscopic (FE-
SEM) studies have been performed in different pHs (pH 7.46,
logical studies of hydrogels have been performed under the
application and release of shear stress in a cyclic manner.
Hydrogels prepared at different pHs are applied a shear stress
of 0.01 to 150 Pa for 6.25 minutes to transform into the cor-
responding sol state and then the recovery of the gel from the
sol state has been studied at 0.01 Pa stress for 10 minutes.
Fig. 5 shows the ow and recovery of the hydrogel prepared at
pH 7.46. During the application of a stress value, the hydrogel
is transformed into a sol within one minute. Aer removal of
8.5, 9.5, 10.0, 10.5, 11.0, 12.0, 13.0 and 14) at MGC. From the FE-
SEM studies, it has been observed that the gelator molecules are
self-assembled into a nanobrillar assembly in the aggregated
gel state (Fig. 6).
0
00
the shear stress, gel formation starts immediately (G > G ).
Here, the gel strength is fully recovered within the time span of
A three-dimensional assembly of gel nanobers has been
observed from the dried hydrogel prepared in phosphate buffer
solution at pH 7.46. These gel nanobers are le-handed helical
in nature (Fig. 6). Widths of these nanobers range from 30 nm
to 55 nm and these nanobers are several microns in length.
High aspect ratios of the gel bers clearly indicate that the
1
0 minutes. It is very interesting to note that the gel strength of
the hydrogel (pH 7.46) is fully recovered up to three consecu-
tive cycles. Moreover, the recovered gel is strengthened with an
increase of the recovery time up to 60 minutes, as shown in
Fig. S14, ESI.† However, pH has a signicant role on the
thixotropic property of the hydrogel. The recovery property is
signicantly affected by the increase in pH. The gel strength is
not fully recovered at higher pH values (pH > 7.46).
Fig. 5 Demonstration of thixotropic property of the hydrogel material prepared
at pH 7.46. Variation of storage modulus and loss modulus have been studied
under application and release of shear stress in a cyclic way.
Fig. 6 FE-SEM images of the hydrogels at different pHs showing a pH-depen-
dent change in morphology.
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intermolecular interactions between the gelator molecules are
6
j
highly anisotropic. However, at higher pH (pH 14), the gel
nanobers exhibit a different type of morphology i.e. tape-like
nanobers. These bers range from 80 nm to 210 nm in width.
Widths of these gel nanobers in aqueous solution of pH 14 are
wider than that of pH 7.46. It has also been observed that at
nearly neutral to slightly basic pH (7.46–11.0) the gel bers are
helical in nature. However, at higher pHs ($12.0) non-helical,
straight, tape-like morphologies have been observed. Gel bers
obtained from the dried hydrogel at pH 12.0 are also wider than
gel bers obtained from the pH range 7.46–11.0 (Fig. 6).
Branching in gel-nanobers starts from this pH (pH 12.0) and
this morphological feature has been observed up to pH 14.0.
These nanobers are originated from a focal point, creating
branched bers. It can be said that the pH has a marked
impact on the nanoscale morphology of the self-assembled gel
phase material along with the gel strength.
In a phosphate buffer solution of pH 7.46, the hydrogel 7.46), 1401 cm (pH 8.5) and 1411 cm (pH 14) in the cor-
nanobers are exible and relatively thinner in width and these responding dried gel states due to the formation of a carbox-
gel nanobers are not laterally overlapped like the nanobers ylate anion at the mentioned pHs. These mentioned peaks are
obtained from the dried hydrogels derived at pH 14. So, a due to the symmetric stretching frequency of the carboxylate
Fig. 7 FT-IR spectra of the dried hydrogels at different pHs as indicated.
14
ꢁ
1
ꢁ1
20c
distinct morphological change from helical nanobers to tape- group of gelator molecules. This observation suggests that in
14
like nanobers has been noticed. This indicates the higher the gel state, the gelator molecules are present in carboxylate
ꢁ
order assemblies in nanoscale of gelator molecules in water are (–COO ) form. For a dried hydrogel at pH 7.46, the pC]O
different in different pHs. At a relatively lower pH range (pH stretching peak corresponding to the amide carbonyl appeared
ꢁ
1
7
.46–11.0), the assemblies of gelator molecules favour helical at 1625 and 1647 cm and at pH 8.5, this type of pC]O
ꢁ
1
structures, which are transformed into a tape-like structure with stretching peak appeared at 1627 and 1648 cm . However,
an increase in pH. The exact reason for this morphological this pC]O stretching peak of dried hydrogel prepared at pH
ꢁ
1
transformation is not clear to us and it is yet to be explored. The 14 appeared at 1644 cm . Moreover, a similar trend of amide
morphology of the hydrogel has also been studied aer the gel carbonyl stretching frequency for the gelator in different wet
recovery process to examine whether the gel nanobrillar gel states has been noticed (Fig. S23, ESI†). N–H stretching
morphology is retained or not. Gel nanobers have been found frequencies of the gelator appeared at 3410, 3411 and 3434
ꢁ
1
to be in the le-handed helical orientation even aer the gel cm
for the dried hydrogels obtained from the aqueous
solutions of pH 7.46, 8.5 and 14 respectively. Hence, the
recovery process (Fig. S20, ESI†).
The morphology of this hydrogel prepared using phosphate hydrogen-bonding interaction plays an important role in gel
buffer (pH 7.46) in a wet gel state has also been studied formation. Though in all these three cases, hydrogelation is
(
Fig. S21, ESI†) by a uorescence microscope in 40ꢂ magni- achieved in the corresponding carboxylate form of the gelator
cation. A piece of wet gel was placed upon the cover slip and it molecules, there is a substantial change in the molecular
has been subjected to a uorescence microscopic study. Due to orientations of the gelator molecules in their respective gel
the presence of a chromophoric pyrene moiety in the gelator states as it is evident from the FT-IR results. This is due to the
(Pyr 1), bluish colored gel bers have been noticed (Fig. S21, change of pH and the variation of the ionic strength of the
ESI†). This indicates the formation of gel-bers upon gelation in aqueous solution.
the wet gel state.
A uorescence spectroscopic study is an important tool to
study the interactions among the gelator molecules in the gel
state. Our gelator molecule contains a chromophoric pyrene
residue. Pyrene is an excellent moiety to study the uorescence as
Structural study
Fourier transform infrared spectroscopy (FT-IR) is a powerful its emission changes due to the variation of environments. A
tool for studying non-covalent interactions, like hydrogen uoresce spectroscopic study of the gelator has been performed
3o,4g,20
bonding, in the self-assembly of gelators.
In the solid in the gel state at different pHs 7.46 and 14 to monitor the
state of Pyr 1, a characteristic peak pC]O stretching frequency emission property of the pyrene moiety. Fig. 8 shows the uo-
corresponding to the carboxylic acid (–COOH) has been rescence spectra of the hydrogelator in two different pHs
ꢁ
1
observed at 1703 cm (Fig. S22, ESI†), whereas, in the dried (pH 7.46 and pH 14). At pH 7.46, there are two main emission
ꢁ1
hydrogel state, no such peak at 1703 cm has been noticed peaks at 398 nm and 489 nm, corresponding to the signals of the
Fig. 7). In the gel state, the carboxylic acid part of the gelator monomer and the excimer of the pyrene moiety. However, at pH
(
molecule is deprotonated to form the carboxylate anion. So, 14, the emission prole changes considerably. The extent of the
the stretching frequency of the pC]O band of carboxylic acid monomer pyrene signal has reduced signicantly with the
ꢁ1
ꢁ1
is shied from 1703 cm in the solid state to 1402 cm (pH concomitant formation of excimers to a great extent. This
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Circular dichroism (CD) spectroscopy is a powerful tech-
12c,i,l,14,22
nique for studying molecular self-assembly.
A CD study
of the gelator has been done at its minimum gelation
concentration at different pH values. In phosphate buffer
solution 7.46, two peaks with a positive cotton effect have been
observed, centered at 365 nm and 301 nm respectively (Fig. 10).
However, the ellipticity of the gel gradually diminished
signicantly with a slight blue shi with an increase in the pH
value. At a higher range of basic pH ($12.0), no apparent CD
signal has been observed and it is almost like a baseline
(Fig. 10). Diminishing of the CD signals at highly basic pH can
be correlated with the pH dependent morphological trans-
formation from chiral nanober (helical) to achiral (straight)
gel nanobers. This suggests that the orientation of gelator
molecules in lower pH range (7.46–11.0) favours the formation
of a chiral (helical) structure and the almost disappearance of
the CD signal at higher pH ($12.0) favours the arrangement
Fig. 8 Fluorescence spectra of the gelator in different pHs. Both spectra indicate
the formation of excimers.
that supports
morphology.
a
non-helical achiral straight tape-like
observation suggests that the uorescence pattern of the pyrene
group is changed due to the alteration of the environment
around the pyrene moiety, such as pH and the ionic strength of
the medium. So, it can be stated that the pH dependent uo-
Encapsulation and sustained release of drugs
rescence pattern of the gelator may be due to the different Most of the previous studies for the encapsulation of biomol-
packing arrangements of gelator molecules in two different pH ecules within the hydrogel matrix require a hot aqueous
values.
X-ray powder diffraction (XRD) of the dried hydrogel mate- cules have been dissolved and upon cooling the drug con-
solution of the gelator molecule, in which desired biomole-
23
rials has also been performed and this provides information taining hydrogels has been obtained. However, the hot
regarding the molecular pattern in their self-assembled gel conditions may cause the diminishing/abolishing of the
state. The XRD patterns of the dried hydrogels at different pHs drug's/biomolecule's activity. In this study, bioactive molecules
ꢀ
˚
are plotted in Fig. 9. A peak at 2q ¼ 26 with a d value 3.4 A can be successfully encapsulated within the hydrogel matrix
appeared and this is a characteristic peak for the aromatic– utilizing the thixotropic property of this amino acid based
3p
aromatic interaction. However, the overall XRD peaks of dried hydrogel (Pyr 1) at pH 7.46. The encapsulation of vitamin B12
hydrogels in phosphate buffer at pH 7.46 and pH 8.5 are similar in the hydrogel matrix has nicely illustrated in Fig. S24 (ESI†).
and this pattern is signicantly different from that of the dried Vitamin B12 and a potent anticancer drug doxorubicin have
hydrogel obtained at higher pH (pH 14). Different XRD patterns been encapsulated within the hydrogel. Vitamin B12 belongs to
have appeared due to the formation of different structures that the class of water-soluble vitamins and it has a vital role in the
result from the change in pH and ionic strength of the gel normal functioning of brain and nerves. It is also required for
medium. This supports the different morphological features of the normal metabolism of cells. Doxorubicin is a well-known
these nanobers in different pHs. A previous report suggests anticancer drug against advanced breast cancer and it also
that this type of morphological transformation is triggered by provides good results against gastric carcinoma.24
sonication in a peptide based assembled system and is sup-
21
ported by different XRD patterns.
Fig. 9 Wide angle X-ray powder diffraction patterns of the dried hydrogels at
different pHs suggesting the changes of the XRD patterns with varying pHs.
Fig. 10 Circular dichroism (CD) spectra of the hydrogels at different pHs. The CD
signal varied upon varying the pH of the medium.
Soft Matter
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Initially, the hydrogel was prepared in a glass vial by dis-
solving the gelator in the phosphate buffer solution of pH
.46. The hydrogel obtained aer cooling for a few minutes A pyrene conjugated protein amino acid based superhydrogel
Conclusions
7
has been transformed into a sol by vortexing. The aqueous has been discovered. This amino acid derivative has been found
solution of vitamin B12 and doxorubicin was added to the sol to form a hydrogel in a wide range of aqueous solutions of pH
(
broken gel) separately. The mixture has been homogenized (7.46–14). Interestingly, it shows a pH-dependent thixotropic
well by vortexing again. Then the resulting solution has been property and morphological transformation. distinct
A
le undisturbed for about an hour to achieve a vitamin B12 or morphological change from le-handed helical nanobers to
doxorubicin entrapped self-supported hydrogel. Interestingly, tape like nanobers has been noticed with an increase of pH of
in this procedure no heating is required for encapsulation. the aqueous medium. The morphological changes of gel
The drug/vitamin encapsulated gel material has been covered nanobers with an increase of pH value have been correlated
by a phosphate buffer solution of pH 7.46 and placed inside a with the changes in the XRD pattern and CD signals. This
ꢀ
37
C incubator. A small aliquot of the buffer solution has suggests the self-assembly of gelator molecules in a lower pH
been taken at different time intervals and it has been sub- range (7.46–11.0) favours the formation of a chiral (le handed
jected to UV/visible spectroscopic studies at 525 nm and 490 helical) structure and the almost disappearance of the CD signal
nm for the release of vitamin B12 and doxorubicin respectively. at higher pH ($12.0) favours the arrangement that supports a
The diffusion coefficient of the drug molecule has been non-helical achiral, straight tape-like morphology. The change
determined on the basis of a non-steady state diffusion model of pH causes an alteration in the nanoscale morphology as well
equation
as the macroscale property (thixotropy) in different pH values.
The thixotropic property of the hydrogel has been successfully
utilized for the encapsulation of biomolecules (vitamin B12 and
anticancer drug doxorubicin) at room temperature without any
heating–cooling cycles and their sustained release to the
surroundings has been studied at physiological pH (7.46) and
2
1/2
(
M
t
/M
a
) ¼ 4(Dt/pl )
t
where M is the total amount of drug released during the
measurement, Ma is the total amount of drug that was kept
within the gel matrix, l represents the hydrogel thickness, t is
the time of the measurement, and D is the diffusion constant
of the drug molecule. Interestingly, these hydrogels are stable in
the presence of phosphate buffer in a vial over a period of 70
hours without disruption and aer that the gel started breaking.
From the release study (Fig. 11), the release of vitamin B12 and
doxorubicin from the gel matrices has been observed up to 75%
and 37% respectively up to 3 days. The diffusion coefficients for
the release of vitamin B12 and doxorubicin have been calculated
ꢀ
temperature (37 C) for a period of 3 days. This single amino
acid based pyrene conjugated new so material and its pH
dependent property hold future promise in the development of
functional so-biomaterials.
Experimental section
Materials
Pyrene 1-butyric acid, L-phenylalanine, L-valine, DCC and HOBt
were purchased from Sigma Aldrich. Solvents for purication
were purchased from SRL, India.
ꢁ
11
2
ꢁ1
ꢁ11
2
ꢁ1
from Fig. 11 as 4.38 ꢂ 10
m s and 1.07 ꢂ 10
m s for
vitamin B12 and doxorubicin respectively. So, in this study, the
encapsulation of biomolecules at room temperature and the
sustained release of these biomolecules at physiological pH
Synthesis of gelator
ꢀ
(7.46) and temperature (37 C) may have a future application in
These amino acid derivatives were synthesized by conventional
solution phase methods using a racemization free fragment
condensation strategy. The C-terminus was protected as a
drug delivery systems. The thixotropic property of a supramo-
lecular, biocompatible hydrogel at physiological pH and
temperature may be expanded as a future application for a drug
delivery vehicle.
0
methyl ester. Couplings were mediated by N,N -dicyclohexyl-
carbodiimide–1-hydroxybenzotriazole (DCC–HOBt). The C-
terminal methyl ester group was removed using 1 M NaOH
solution. The intermediate compound was puried by column
chromatography using silica gel (100–200 mesh size) as a
stationary phase and chloroform and ethyl acetate as the eluent.
1
13
Puried compounds were also characterized by H NMR,
C
NMR and HRMS spectroscopy.
Pyrene-Phe-OMe
Pyrene 1-butyric acid (2.88 g, 10 mmol) in DMF (8 mL) was
cooled in an ice-water bath. H-Phe-OMe was isolated from the
corresponding methyl ester hydrochloride (4.33 g, 20 mmol) by
neutralization, subsequent extraction with ethyl acetate and
concentration to 10 mL. Then it was added to the reaction
mixture, followed immediately by DCC (2.06 g, 10 mmol) and
Fig. 11 Release profile diagram of doxorubicin and vitamin B12 from the Pyr 1
based hydrogel prepared at phosphate buffer solution at pH 7.46.
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HOBt (1.35 g, 10 mmol). The reaction mixture was stirred for (401.1991): C, 77.78; H, 6.78; N, 3.49; found C, 77.76; H, 6.80; N,
+
+
three days. The reaction mixture was taken in ethyl acetate 3.50%. HRMS (m/z): 402.2098 [M + H] , 424.1875 [M + Na] ,
(
+
+
+
60 mL) and the DCU (dicyclohexylurea) was ltered off. The 425.1963 [M + Na + H] , 440.1679 [M + K] , 825.3402 [2M + Na] .
organic layer was washed with 1 M HCl (3 ꢂ 50 mL), brine (2 ꢂ
5
0 mL), 1 M sodium carbonate (3 ꢂ 50 mL), and brine (2 ꢂ 50 Pyr 2 (pyrene-Val-OH)
mL) and then dried over anhydrous sodium sulphate and
evaporated in vacuum to yield the peptide as a white solid.
Purication was done by a silica gel column (100–200 mesh)
1
ꢀ
Yield: H NMR (300 MHz, DMSO-D , 25 C, d): 12.55 (1H, br.),
6
8
7
1
2
1
1
.37 (1H, d, J ¼ 9.3), 8.28–8.18 (4H, m), 8.12–8.03 (4H, m),
.93(1H, d, J ¼ 7.8), 4.22–4.17 (1H, m), 2.36–2.26 (2H, m), 2.11–
using chloroform and ethyl acetate as the eluent.
13
.96 (3H, m), 0.91–0.84 (6H, m). C NMR (75 MHz, DMSO-D
6
,
1
Yield: 2.48 g (6.79 mmol, 67.90%). H NMR (300 MHz, CDCl ,
ꢀ
3
5 C, d): 173.8, 172.9, 137.2, 131.5, 131.0, 129.9, 128.7, 128.1,
28.0, 127.8, 127.1, 126.7, 125.5, 125.5, 125.3, 124.8, 124.7,
24.1, 57.8, 35.3, 32.8, 30.3, 28.3, 19.8, 18.7. Anal. calc. for
ꢀ
TMS, 25 C, d): 8.28 (1H, d, J ¼ 9.3), 8.18–7.97 (7H, m), 7.83 (1H,
d, J ¼ 7.8), 5.86 (1H, d, J ¼ 7.3), 4.97–4.91 (1H, m), 3.73 (3H, s),
3
2
1
1
1
.37–3.32 (2H, m), 3.21–3.05 (2H, m), 2.23–2.28 (2H, m), 2.20–
.15 (2H, m). C NMR (75 MHz, CDCl
72.28, 135.97, 135.88, 131.55, 131.05, 130.08, 129.35, 128.89,
28.73, 127.61, 127.52, 127.28, 126.84, 125.96, 125.12, 125.03,
24.91, 123.52, 53.12, 38.06, 35.93, 32.79, 27.31. Anal. calc. for
C H NO (387.18834): C, 77.49; H, 6.50; N, 3.61; found C,
2
5
25
3
1
3
ꢀ
3
, TMS, 25 C, d): 172.32,
+
7
4
7.50; H, 6.48; N, 3.59%. HRMS (m/z): 409.9499 [M + Na] ,
10.9635 [M + Na + H] , 425.9176 [M + K] .
+
+
Hydrogel preparation
C H NO (449.1991): C, 80.15; H, 6.05; N, 3.12; found C, 80.18;
H, 6.04; N, 3.13%. HRMS (m/z): 450.0418 [M + H] , 472.0214 [M +
Na] , 487.9909 [M + K] .
3
0
27
3
+
Hydrogels were prepared by heating the corresponding gelator
in the aqueous solutions of different pH in glass vials (inner
diameter 1.1 cm) till the gelator dissolved. Upon cooling at
room temperature for 1 minute, self-supported hydrogels were
obtained.
+
+
Pyr 1 (pyrene-Phe-OH)
Pyrene-Phe-OMe (3.5 g, 7.04 mmol) was dissolved in MeOH
(25 mL) and 1 M NaOH (10 mL) was added. The reaction mixture
Instrumentations
was then stirred and the progress of saponication was moni-
tored by thin layer chromatography (TLC). Aer 10 h methanol
was removed under vacuum, the residue was taken in 50 mL
water and washed with diethyl ether (2 ꢂ 50 mL). Then the pH
of the aqueous layer was adjusted to 2.0 using 1 M HCl and it
was extracted with ethyl acetate (3 ꢂ 50 mL). The extracts were
pooled, dried over anhydrous sodium sulphate and evaporated
in vacuum to yield as a white solid.
Field-emission scanning electron microscopy (FE-SEM). For
FE-SEM studies, hydrogels were prepared in different pH solu-
tions at a xed concentration (0.04% w/v) and all these prepared
ꢀ
hydrogels were aged for 25 minutes at 27 C before SEM sample
preparation. FE-SEM experiments were studied by placing a
small portion of the gel samples on a microscope cover glass.
Then, these samples were dried rst in air and then in vacuum
and coated with platinum for 90 seconds at 10 kV voltages and
10 mA current. The average thickness of the coating layer of
platinum was 3 to 4 nm. Aer that micrographs were taken
using a SEM apparatus; Jeol Scanning Microscope-JSM-6700F.
Fluorescence microscopy. The native gel-phase material was
placed on a glass microscope slide and examined under a
1
Yield: 3.1 g (6.45 mmol, 91.6%). H NMR (300 MHz, DMSO-
ꢀ
D
6
, 25 C, d): 12.70 (1H, s), 8.34–8.06 (9H, m), 7.87 (1H, d, J ¼
7
3
1
1
1
3
7
.8), 7.26–7.15 (5H, m), 4.52–4.48 (1H, m), 3.21–3.18 (2H, m),
.13–3.07 (1H, m), 2.91–2.83 (1H, m), 2.23–2.21 (2H, m), 1.94–
.92 (2H, m). C NMR (75 MHz, DMSO-D , 25 C, d): 173.1,
71.9, 137.7, 136.5, 130.8, 130.4, 129.2, 128.9, 128.0, 127.4,
27.1, 126.4, 126.3, 126.0, 124.8, 124.7, 124.2, 124.1, 123.5, 53.3,
9.1, 36.7, 34.7, 27.4. Anal. calc. for C29
1
3
ꢀ
6

uorescence microscope (OLIMPUS BX-61) in 40ꢂ
magnication.
3
H25NO (435.1834): C,
FT-IR spectroscopy. FT-IR spectra of the dried gel were
recorded using a KBr pellet technique by a Nicolet 380 FT-IR
spectrophotometer (Thermo Scientic). Dried gel materials of
9.98; H, 5.79; N, 3.22; found C, 79.95; H, 5.82; N, 3.24%. HRMS
m/z): 436.0821 [M + H] , 458.0478 [M + Na] .
+
+
(
different pHs were dissolved in D
and the FT-IR of these wet gels were studied using a CaF
Differential scanning calorimetry (DSC). The gel melting
temperature, and other thermal behaviours were measured
using a Perkin-Elmer differential scanning calorimeter (Dia-
2
O to get the wet gel materials,
cell.
Synthesis of Pyr 2
2
The synthetic procedure of Pyr 2 is similar to the method of
Pyr 1.
Pyrene-Val-OMe
mond DSC-7) working under a N
2
atmosphere. Gel samples
1
ꢀ
H NMR (300 MHz, CDCl
3
, TMS, 25 C, d): 8.22 (1H, d, J ¼ 9.2), were taken in large volume capsules (LVC) tightly sealed with an
ꢀ
ꢀ
8
5
.09–8.01 (4H, m), 7.94–7.88 (3H, m), 7.78 (1H, d, J ¼ 7.8), 5.84– O-ring. These samples were then heated from 25 C to 85 C at
ꢀ
ꢁ1
.81 (1H, d, J ¼ 8.3), 4.56–4.51 (1H, m), 3.65 (3H, s), 3.34–3.29 the heating rate of 5 C min . Cooling runs were also taken
ꢀ
ꢀ
(
2H, m), 2.31–2.26 (2H, m), 2.19–2.04 (3H, m), 0.87–0.80 (6H, aer waiting 10 min at 85 C and then cooled at the rate 2 C
1
3
ꢀ
ꢁ1
ꢀ
m). C NMR (75 MHz, CDCl , TMS, 25 C, d): 172.8, 172.6, min to 25 C.
3
1
1
3
35.9, 131.6, 131.0, 130.1, 128.9, 127.6, 127.5, 127.5, 126.8,
25.9, 125.2, 125.1, 125.0, 124.89, 123.50, 57.08, 52.23, 36.10, hydrogels was carried out by placing the hydrogels prepared at
X-ray diffraction study. An X-ray diffraction study of the dried
2.86, 31.42, 27.48, 19.07, 18.00. Anal. calc. for C26
H
27NO
3
different pHs on glass plates. These hydrogels are allowed to dry
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Soft Matter
at room temperature for 3 days. Experiments were then carried the anonymous reviewers for their very helpful comments for
out using an X-ray diffractometer (Bruker AXS, Model no. D8 the improvement of manuscript. Jayanta is also thankful to his
Advance). The instrument was operated at 40 kV voltages and two friends for their generous help and valuable comments
40 mA current using Ni-ltered CuKa radiation and the during the preparation of the revised manuscript.
instrument was calibrated with a standard Al (corundum)
2 3
O
sample before use. The LynxEye super speed detector was used
with scan speed 0.3 s and step size 0.02 .
Notes and references
ꢀ
Rheology. Rheological experiments were performed with an
AR 2000 advanced rheometer (TA Instruments) using a cone
plate geometry in a Peltier plate. The cone diameter was 40 mm,
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ꢀ
0
00
the cone angle was 1 59 50 , and the truncation was 56 mm. For
angular frequency sweep experiments, the angular sweep was
ꢁ
1
ꢀ
conducted from 10 to 100 rad s at a xed strain 0.01% at 27 C
temperature. The thixotropic property was conducted to
examine the alteration of rheological properties of hydrogel
material under the application and the release of shear stress.
This process includes two steps: (i) deformation of the hydrogel
with increasing shear stress from 0.01 to 150 Pa for 6.25
minutes and (ii) recovery of sol state into gel state under low
shear stress (0.01 Pa) for 10 minutes. In these two steps, the
frequency was xed at 1 Hz. Four consecutive cycles were
applied on the hydrogels to compare the extent of gel strength
recovery.
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ꢁ1
formed by purging dry N gas continuously at 15 L min during
2
data acquisition. Data were collected in a quartz cuvette with a
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was prepared in a glass vial by dissolving the gelator in the
phosphate buffer solution of pH 7.46. The hydrogel obtained
aer cooling for a few minutes was transformed into sol by
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ꢀ
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Acknowledgements
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
nancial assistance. We also acknowledge the support by
the DST, India, project no.SR/S1/OC-73/2009. We are thankful to
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