Stimuli responsive hydrogels derived from a renewable resource: synthesis, self-assembly in water and application in drug delivery

Article abstract of DOI:10.1039/c5tb00864f

We designed and synthesised coumarin-tris derivatives from a renewable resource and well characterized them using different spectral techniques. The self-assembly of coumarin-tris amphiphiles into hydrogels was examined relative to the molecular structure

Full text of DOI:10.1039/c5tb00864f

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Stimuli Responsive hydrogel derived from Renewable Resource:
Synthesis, Self-Assembly in water and application in drug delivery
Krishnamoorthy Lalitha,a Y. Siva Prasad,a C. Uma Maheswari,a Vellaisamy Sridharan,a George
John,b Subbiah Nagarajan*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We designed and synthesised coumarin-tris derivatives from renewable resource and well characterized using different
spectral techniques. Self-assembly of coumarin-tris amphiphile into hydrogel were examined relative to the molecular
structure. The reversible morphological transition from nanofibers to vesicles and nanotubes has been observed, upon pH
variation. Reversible processes and self-assembled structures such as gel, vesicle and nanotube formation have been
investigated by optical microscopy and High Resolution Transmission Electron Microscopy (HRTEM). 1H NMR and XRD
studies clearly suggest the π-π stacking interaction and hydrogen bonding were the driving force for the process of
gelation. The flow behaviour of hydrogel has been identified using rheological measurements. More importantly,
chemopreventive drug, curcumin has been encapsulated into the gel and further release has been achieved by gel-to-sol
transition induced by pH and metal ion, Fe3+ stimuli. Reported hydrogel could play a substantial role in the development
www.rsc.org/
of
new
generation
stimuli
responsive
drug
delivery
systems
for
in
vivo
formulations
preciously defined biomimetic structures (helices, sheets,
nanotubes, vesicles, micelles, etc.) through non-covalent
Introduction
interactions such as hydrogen bonding,
ꢀ-ꢀ interaction,
Natural systems are having the ability to change its
physiological functions in direct response with environmental
conditions such as temperature, light, pH, electro/magnetic
field, mechanical stress and chemical stimuli has always been
an interesting source of inspiration for scientists to develop
new technologies for advanced “smart” materials.1,2
Remarkable changes in micro/nanoscopic (molecular level) and
macroscopic (surface level) are responsible for stimuli-
responsive behaviour of natural systems. Among the different
types of stimuli-responsive materials, gels are considered as
assuring materials for the bottom-up nanofabrication tools in
various fields such as biology, biomedicine, tissue engineering,
drug delivery, sensors, catalysis, cosmetics, and as
therapeutics.2 Gels are classified in to two categories: (1)
chemical gel and (2) physical gel. Usually chemical gels are
covalently cross-linked irreversible structures (gels derived
from polymers), whereas physical gels, are self-assembled
structures based on reversible intermolecular interactions.3
Depending up on the molecular structure, gels can acquire
certain prescribed conformations and self-assemble into
electrostatic and van der Waals interactions, hydrophilic-
lipophilic balance (HLB) and other supramolecular weak
forces.4 Such self-assembled soft materials are continuous in
structure and own solid-like in rheological behaviour, which is
due to the entrapment of solvent in self-assembled 3D solid
matrix through surface tension and capillary forces.4 In
particular, supramolecular hydrogels derived from small
molecules are more interesting because of its inherent and
excellent biocompatibility and they were considered as best
alternative for chemical gel (polymeric gel). Due to the
dynamic reversible nature of hydrogel derived from small
molecules, the phase transition either sol-to-gel or gel-to-sol
can be triggered by the external stimuli.5 This phenomenon
could be useful for understanding the gelation mechanism and
advanced applications like drug delivery and switchable
devices with memory function. We report herein the ability of
small amphiphilic system bearing a biologically interesting
coumarin moiety to form stable supramolecular hydrogel that
are responsive to external stimuli, pH and biologically
important Fe3+ ion. In particular, we have synthesised and
studied the self-assembly (hydrogelation) and stimuli
responsive behaviour of coumarin coupled tris derivatives,
which in turn derived from renewable plant-derived resource,
cashew nut shell liquid. Renewable resources has been
substantial focus on establishing and optimizing efficient
materials and production of fine chemicals and fuels that
address the needs of the 21st century.6 Cardanol, a bio based
non-isoprene lipid directly incurred by distillation of cashew
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nutshell liquid (CNSL), an important by-product obtained from ethylacetate, dried under Na2SO4 and pure product was
the cashew nut industry. Cardanol comprising of rich mixture isolated by column chromatography using 95:5 v/v hexane-
of phenolic lipids: 5% of 3-n-pentadecylphenol (3-PDP), 50% of ethyl acetate as eluent.
3-(8Z-pentadecenyl)phenol,
16%
29%
of
of
3-(8Z,11Z-
Compound 2b
pentadecadienyl)phenol
and
3-(8Z,11Z,14-
1
pentadecatrienyl)phenol. The uniqueness of cardanol stem Isolated as yellow liquid; yield =88%. H NMR (300MHz, CDCl3)
from its structural lineament and easily accessible saturated δ = 0.88 (t, J = 7.2 Hz, 3H), 1.25-1.30 (m, 16H), 1.59-1.64 (m,
and unsaturated hydrocarbon chains.7
4H), 1.95-2.05 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 5.33-5.39
(m,2H), 6.80 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 7.44 (d, J =7.8 Hz,
1H), 9.83 (s, 1H), 11.05 (s, 1H); 13C NMR (75MHz, CDCl3) δ =
195.8, 161.8, 153.8, 133.6, 130.0, 129.7, 120.5, 118.9, 117.1,
36.4, 32.6, 31.8, 30.7, 29.7, 29.7, 29.7, 29.6, 29.4, 29.3, 29.3,
29.2, 29.2, 29.0, 27.2, 27.2, 22. 7, 14.1.
Experimental
General
All reagents and solvents were purchased from Sigma Aldrich,
Merck, Alfa Aesar and Avra chemicals and were used as such
for the synthesis without further purification. The progress of
the reactions were monitored by thin-layer chromatography
(TLC) using pre-coated silica gel plates purchased from Merck
and visualized under UV cabinet and/or p-anisaldehyde stain
and/or molecular iodine. We have used LR grade solvents for
the compounds purification and AR grade solvents for gelation
studies. If not indicated particularly, all other reagents were
purchased from commercial source and used without further
purification. Silica Gel (100-200 mesh) purchased from Avra
synthesis, INDIA was used for column chromatography. 1H-
and 13C-NMR spectra were recorded in either CDCl3 or CDCl3
with few drops of DMSO-d6 at room temperature on a Bruker
DRX 300 MHz instrument. Chemical shifts (δ) are reported in
parts per million (ppm) with respect to internal standard TMS
and coupling constants (J) are given in Hz. Proton multiplicity is
assigned using the following abbreviations: singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m). MM2 energy
minimised diagram was performed using ChemBio 3D Ultra 13.
Red colour dotted line shows the possible hydrogen bond
formation.
Compound 2c
Isolated as white solid; Yield = 92%. 1H NMR (300MHz, CDCl3) δ
0.88 (t, J = 6.9 Hz, 3H), 1.29-1.34 (m, 24H), 1.57-1.66 (m,2H),
2.61 (t, J = 7.8 Hz, 2H), 6.80 (s, 1H), 6.83 (dd, J = 7.8, 1.5 Hz,
1H), 7.45 (d, J = 7.8 Hz, 1H), 9.83 (s, 1H), 11.05 (s, 1H); 13C NMR
(75MHz, CDCl3) δ= 195.8, 161.8, 153.8, 133.6, 120.5, 118.8,
117.1, 36.5, 31.9, 30.7, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 22.7,
14.1.
General procedure for synthesis of compounds 3b and 3c
To a solution of 2-hydroxyarylaldehyde (2a-c) (3 mmol) and
diethyl malonate (3.2 mmol) in ethanol (15mL), piperidine (0.1
mL) and glacial acetic acid (1 or 2 drop) were added and the
reaction mixture was refluxed for 6h. After completion of
reaction as identified using TLC, 20 mL of water was then
added to the reaction mixture. Crude product was extracted
using ethyl acetate and dried over anhydrous Na2SO4. Pure
product was isolated by column chromatography using 75:25
v/v hexane-ethylacetate as eluent.
Compound 3b
Isolated as yellow liquid; Yield = 87%. ¹H NMR (CDCl3,
300MHz): δ 0.81 (t, J = 6.9 Hz, 3H), 1.19-1.24 (m, 18H), 1.34 (t,
J = 6.9 Hz, 3H), 1.5-1.62 (m, 2H), 1.90-1.95 (m, 2H), 2.65 (t, J =
7.5 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 5.27-5.31 (m, 2H), 7.07 (s,
1H), 7.08 (d, J = 6.9 Hz, 1H), 7.43 (dd, J = 6.9, 1.5 Hz, 1H), 8.45
(s, 1H); 13C NMR (CDCl3, 75MHz) δ = 166.6, 163.3, 157.0, 155.4,
151.3, 148.7, 129.9, 129.6, 129.2, 125.5, 116.9, 116.2, 115.7,
61.8, 61.5, 41.7, 36.7, 30.8, 29.7, 29.7, 29.3, 29.2, 29.1, 28.9,
27.2, 27.1, 22.6, 14.2.
Distillation of Cardanol
The key constituent of CNSL is cardanol 1b, a bio based non
isoprene lipid, consisting of phenolic lipid mixture: 5% of 3-n-
pentadecylphenol (3-PDP), 50% of 3-(8Z-pentadecenyl) phenol,
16% of 3-(8Z, 11Z-pentadecadienyl) phenol and 29% of 3-
(8Z,11Z,14-pentadecatrienyl) phenol. CNSL was distilled at a
temperature between 210 and 280°C, under a pressure from 2
to 8 mm Hg to get cardanol. Cardanol was obtained as pale
yellow liquid which darkens during storage. After a second
distillation, mixture of cardanol mono-, di- and tri-ene was
obtained.
Compound 3c
Isolated as white solid; Yield = 89%. ¹H NMR (CDCl₃, 300MHz):
δ 0.83 (t, J = 6.9 Hz, 3H), 1.18-1.23 (m, 26H), 1.34 (t, J = 6.9 Hz,
3H), 2.65 (t, J = 7.8 Hz, 2H), 4.30, (t, J = 7.2 Hz, 2H), 7.09 (d, J =
6.9 Hz, 1H), 7.20 (s, 1H), 7.44 (dd, J = 6.9, 1.5 Hz, 1H), 8.45 (s,
1H); 13C NMR (CDCl3, 75MHz) δ = 163.3, 1571, 155.4, 151.3,
148.7, 129.2, 125.5, 116.2, 115.7, 61.8, 36.3, 31.9, 30.8, 29.7,
29.7, 29.6, 29.5, 29.4, 29.4, 29.2, 22.7, 14.2, 14.1.
Synthesis
General procedure for synthesis of compounds 2b and 2c.
Dry paraformaldehyde (35 mmol) was added to a mixture of 3-
alkyl phenol (4 mmol), anhydrous magnesium chloride (6
mmol) and triethylamine (15 mmol) in acetonitrile (25 mL) and
the mixture was heated under reflux for about 12-15h. After
the completion of the reaction as monitored by TLC, the
reaction mixture was cooled to room temperature and 5% aq.
HCl was added. The crude product was extracted with
General procedure for synthesis of compound 4a-c
Ethyl 7-alkylcoumarin-3-carboxlate (3a-3c) (1 mmol) and tris
(0.12 g, 1 mmol) (1:1 ratio) in methanol were refluxed with
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stirring for about 24h. After completion of reaction as original gel state upon cooling. Gel was prepared in a 5 mL
identified by TLC, the reaction mixture was then cooled to glass vial as described above, the vial was immersed in the oil-
room temperature. The precipitate thus formed was filtered bath ‘upside down’ and the vial is slowly heated. The
and washed several times with cold methanol. Filtrate temperature at which the gel melted down to solution was
obtained was kept under refrigeration for further crop of recorded as Tg.
product. Pure product was obtained by recrystallization in
Self-assembly studies
ethanol.
In a round bottom flask, 2 mg of compound 4c was dissolved in
Compound 4a
200 μL of ethanol and then 5 mL of double distilled water was
Isolated as white solid; Yield = 92%. ¹H NMR (DMSO-d6, added until to get a bluish white tinge. Entire content was
300MHz): δ 3.68 (d, J = 5.4 Hz, 6H), 4.85 (t, J = 5.4 Hz, 3H), 7.46 refluxed for 1h to get a clear solution and then cooled at room
(t, J = 7.5 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.77 (ddd, J = 8.7, 7.2, temperature. Formation of self-assembled fibers has been
1.5 Hz, 1H), 8.0 (dd, J = 7.8, 1.5 Hz, 1H), 8.93 (s, 1H), 8.98 (s, observed within 24h. This experiment has also been carried
1H).
out in both acidic and basic pH levels.
Compound 4b
Morphological analysis
Isolated as pale yellow semi solid; Yield = 79%. ¹H NMR (CDCl₃, Morphological analysis of hydrogel formed by gelator was
300MHz): δ = 0.89 (t, 3H), 1.25-1.32 (m, 14H), 1.66-1.90 studied using Carl Zeiss AXIO ScopeA1 fluorescent/phase
(m,8H), 2.75 (m, 2H), 3.90 (s, 6H), 5.31-5.32 (m, 2H), 7.20-7.28 contrast microscope. A glass slide containing a small portion of
(m, 2H), 7.59 (d, J = 8.4 Hz, 1H), 8.86 (s, 1H), 9.74 (s, 1H); 13C gel was mounted on Phase Contrast Microscope and the
NMR (CDCl3, 75MHz) δ = 162.1, 160.4, 153.7, 153.7, 150.4, morphology of gel was identified. The reversible morphological
147.1, 128.7, 115.9, 115.4, 115.1, 61.7, 61.3, 35.3, 30.7, 29.8, transition from nanofibers to vesicles and nanotubes have
28.7, 28.4, 28.2, 26.2, 21.6, 13.1. HRMS (ESI): calcd for been investigated by using JEOL JEM 2100F FETEM.
C29H43NO6, m/z 502.3169 [M+H]+; found m/z 502.3184.
X-Ray diffraction studies
Compound 4c
A small portion of a wet hydrogel sample was placed on a glass
0
Isolated as white precipitate; Yield = 78%; mp 106-110 C. ¹H slide and allowed to dry. X-ray diffraction pattern of xerogel
NMR (CDCl₃, 300MHz): δ 0.87 (t, J = 6.3 Hz, 3H), 1.25-1.32 (m, thus formed was analyzed on XPert-PRO Diffractometer
24H), 1.64-1.67 (m, 2H), 2.74 (t, J = 7.8 Hz, 2H), 3.74 (d, J = 5.7 system.
Hz, 6H), 4.83-4.85 (m, 3H), 7.17 (d, J = 7.5 Hz, 1H), 7.22 (s, 1H),
7.64-7.66 (m, 1H), 8.88 (s, 1H), 9.53 (s, 1H); 13C NMR (CDCl3,
Fourier Transform Infrared Spectrometer (FTIR)
75MHz) δ = 162.0, 160.7, 154.2, 150.6, 147.8, 129.2, 125.5, Infrared spectrum was recorded using FTIR Shimadzu 8000
116.9, 115., 115.3, 62.3, 61.7, 38.7, 35.7, 31.3, 30.3, 29.1, 29.0, Spectrometer in the spectral range of 4000 cm-1 to 400 cm-1.
28.9, 28.8, 28.7, 28.6, 22.1, 13.7. HRMS (ESI): calcd for Approximately 1.5 mg of sample was ground with 15-25 mg of
C29H45NO6, m/z 504.3325 [M+H]+; found m/z 504.3326.
potassium bromide using pestle and mortar. The fine powder
was then transferred to the ‘dye’ and 10 tonne of pressure was
applied for 2 minutes. The resulting pellet was put in the
Gelation studies
A known quantity of gelator was mixed with appropriate sample holder and the FTIR spectrum was recorded.
amount of solvent in a sealed test tube, and the system was
Rheological measurements
heated until the solid get dissolved. By this procedure the
solvent boiling point becomes higher than that under standard The mechanical properties of hydrogel and curcumin
atmospheric pressure. The resulting solution was slowly encapsulated composite gel were investigated with a stress
allowed to cool to room temperature, and gelation was controlled rheometer (Anton Paar 302 rheometer) equipped
visually observed by inverting the test tube. A gel sample thus with a steel-coated parallel-plate geometry (25 mm diameter).
obtained which exhibited no gravitational flow in inverted tube The gap between two plates was fixed as 1 mm. The
is denoted as “G”. Different solvents and vegetable oils such as measurements were carried out at 23 ̊C. Firstly, amplitude
hazelnut oil, sesame oil, jojoba oil, olive oil, soya bean oil, sweep measurement was conducted, which provides the
linseed oil, heavy paraffin oil, light paraffin oil, dodecanol, information about linear viscoelastic range which is directly
ethanol, n-butanol, octanol, toluene, cyclohexane, n-heptane, proportional to the mechanical strength of the gel sample.
ethylene glycol, and mixture of solvents such as DMSO, DMF, Secondly, the storage modulus, G’ and the loss modulus, G”
DMSO-water, DMF-water were used for gelation studies and were monitored as functions of frequency from 0.1 to 100 rad
results were given in ESI.
s-1.
Gel-sol melting temperature (Tg)
Preparation of curcumin encapsulated hydrogel (composite gel)
Gel melting temperature was determined by flow of gel by test We have used curcumin, one of the anticancer drug occurring
tube inversion method. Above the gelation temperature, the naturally, for drug loading and release studies. The
gel phase becomes solution, but could be returned to their encapsulation of chemopreventive drug curcumin was done by
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adding 5 mg of curcumin into the hydrogel (5mg of 4c in 5mL structures.8 In fact hydrogel formed by 4c display a gel to sol
of DMSO-water mixture i.e. 1 % wt/v) followed by heating to transition upon heating-cooling cycles (Tg = 45 C) and Tg
̊
get a clear yellow solution and cooled to room temperature. increases with increase in concentration of gelator. To acquire
The formation of composite gel was confirmed by the perceptivity into the protons involved in the self-assembly
observation of no gravitational flow upon test tube inversion.
process, we have recorded 1H NMR spectra of gelator 4c in
both solution and gel states.
Stimuli responsive drug release studies
pH as stimuli responsive drug release studies: To the
composite gel 10 mL of acidic buffer (pH = 4) was added and
1mL of aliquots was retrieved at different time intervals. On
adding acidic buffer, the simultaneous degradation of
composite gel and release of curcumin drug was visually
observed. The drug release profile was monitored by UV-vis
spectra by diluting 1.0 mL of the retrieved aliquot to 2.0 mL
using double distilled water.
Metal ion, Fe3+ as stimuli responsive drug release studies: To
the composite gel 10 mL of FeCl3 solution of concentration
1X10-3 M was added and 1mL of aliquots was retrieved at
different time intervals. On adding FeCl3 solution, the slow
diffusion of Fe3+ ions with simultaneous degradation of
composite gel and release of curcumin drug was observed. The
drug release profile was monitored by UV-vis spectra by
diluting 1.0 mL of the retrieved aliquot to 2.0 mL using double
distilled water.
CHO
(CH2O)n
TEA, MgCl2,
MeCN, reflux
R
OH
R
OH
1a-c
2a-c
C2H5O
OC2H5
ethanol, piperidine,
AcOH, reflux, 6h
OH
O
O
R
O
O
O
OH
N
H
OC2H5
tris, MeOH
OH
R
O
4a-c
reflux, 12h
O
O
3a-c
(1-4)a R =
H
15:1 (50%)
15:2 (16%)
15:3 (29%)
(1-4)b R =
(1-4)c R =
15:0 (5%)
15:0 (100%)
Scheme 1. Synthesis of coumarin coupled tris amphiphile 4a-c
Results and discussion
The stimuli responsive coumarin-tris derivatives were
synthesised as follows: Electrophilic aromatic substitution
reaction of cardanol, 1b and n-pentadecylphenol (PDP), 1c
with paraformaldehyde in presence of MgCl2 and TEA lead to
the formation of cardanol-aldehyde 2b and PDP-aldehyde 2c
respectively. The Knoevenagel reaction of compounds 2a-c
with diethylmalonate led to the desired ethyl coumarin-3-
carboxylate 3a-c in good yields. Ethylesters 3a-c were
converted into corresponding tris amide (4a-c) under mild
conditions in good yield (Scheme 1). In this report we have
synthesised and completely characterized (NMR and Mass
spectral analysis) three different coumarin-tris compounds
with varying hydrophobicity. Synthetic protocol used for the
construction of amphiphiles are simple, green and economical
for practical applications. Biocompatible tris were introduced
as the hydrophilic part to empower the amphiphile for
excellent biological functions. The gelation ability of compound
4a-c was consistently studied for both hydrophilic and
hydrophobic solvents by “stable to inversion” method.2
Compound 4c exhibit excellent hydrogelation in DMSO-water
by heating, followed by sonication for few seconds. Compound
4c shows critical gelation concentrations (CGCs) of 0.8 % (wt/v)
in DMSO-water (1:2 ratio). Gel thus formed are thermo
reversible, and stable at room temperature even for more
than 5 months. In case of 4a and 4b remarkable stifling of
gelation ability was observed, probable due to the lack of
hydrophobic part in 4a and the existence of a kink in
hydrophobic part of 4b. This result highlight the importance of
maintenance of optimum hydrophilic and hydrophobic
interactions, which driven to assemble into macromolecular
In general a gradual shift or broadening of signals under
1
aggregated/self-assembled state were observed in 1D H NMR
spectrum and folding/unfolding of self-assembled structure
could be identified by 2D NMR spectra.9 In 1D 1H NMR spectra
of 4c dissolved in DMSO-d6, protons in coumarin moiety Ha-Hd
resonates at
in gel form Ha-Hd protons experience downfield shift and
resonate at 8.91, 7.92, 7.38, 7.42 ppm respectively. Note
δ 8.87, 7.65, 7.22, 7.21 ppm respectively, whereas
δ
that for NMR studies, hydrogel was prepared by dissolving 4c
in DMSO-d6 - D2O solvent mixture and in gel form, all –OH and
–NH peaks disappear because of D2O exchange. This result
clearly depicts the existence of π-π stacking in gel state (Figure
1), which was further confirmed by XRD analysis. The stacked
1H NMR spectra of gelator 4c and its gel is given in ESI.
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Figure 1. 1H NMR spectra (aromatic region alone) of (a) gelator, 4c in DMSO-d6 and (b)
gel formed by 4c in DMSO-d6-D2O (1:2 ratio).
hydrophobic moiety. The broad peak at 20º (0.45 nm) is
assignable to the (001) aspect of
moiety (see ESI).10 A schematic representation of gelator
arrangement in gel phase is given in Figure 3b.
π-π stacking of coumarin
In order to obtain further insight into the aggregation mode at
nanoscale we performed optical microscopy and FE-TEM. A
slice of the gel was carefully taken and subjected for
morphological analysis. The existence of well dispersed
nanofibers in gel was also identified by optical microscopy
(Figure 2a). From HRTEM analysis one can consistently observe
a
well-developed network structure composed of 3D
entangled fibrous network with diameter range of 50-200 nm
(Figure 2b). The existence of nanofiber with greater void
volumes can trap more solvent in it as it is documented from
HRTEM analysis.
Figure 3. (a) SAXD pattern of gel formed by 4c and (b) schematic representation of
possible molecular packing models for the gel.
This gel is highly stable in neutral and basic pH for a long time,
while change in pH from neutral to acidic condition by adding
buffer solution (pH = 4.0) drive the slow conversion of gel to
sol state. This behavior could be attributed due to the
disturbance of H-bonding exerted by amide group. The gel-to-
sol conversion in response with the external stimuli, pH could
be directly visualized by naked eye. Molecular level
commutes11 occurred during this process was identified by
HRTEM analysis. On addition of acidic buffer, fibrillar
morphology of gel are changed to vesicular and nanotubular
structures with average diameter of 300 nm and 50 nm
respectively (Figure 4a & 4b). The average size of the vesicles
was further confirmed using particle size analyzer. The average
size of the vesicles formed by adding acidic buffer (pH = 4) into
the gel, formed by 4c was 243 nm (see ESI). It should be noted
that the vesicles and nanotubes formed by 4c were stable for
even more than 3 months. The observed results indicate that
the molecule self-assemble into fibers in both neutral and
basic medium (as gel form) and vesicles and nanotubes in
acidic medium. This property could be attributed to the H-
bonding triggered self-assembly12 behavior of 4c under
Figure 2. Morphological analysis of gel, 4c. (a) Optical microscopy image of hydrogel
formed by 4c and (b) HRTEM image of xerogel of 4c.
Small angle X-ray diffraction (SAXD) was employed to gain the
additional perceptiveness into the structure that comprise the
gel formed from 4c. XRD of wet gel gave a Bragg’s reflection at
4.02, 1.35, 1.00 and 0.45 nm. The energy minimized structure
of compound 4c indicate the molecular length of 2.74 nm,
which is different from the observed d-spacing value (4.02 nm)
from XRD studies (Figure 3a). The d-spacing value observed is
in between the mono and double layer thickness formed by
the end to end length of 4c, which clearly validate the
molecular self-assembly in an interdigitated fashion of
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different pH solution. Solubilization of hydrophobic drugs curcumin and intensity increases with increase in time (Figure
formulating a suitable delivery system is an ambitious chore in 4e). Metal ion, Fe3+ plays a significant role in biological
biotechnology.13 By getting clue from the self-assembly processes as metalloenzyme and hemeprotein, to name a
behavior of 4c in different pH levels, we gear-up to few.15 It has been reported in literature that coumarin coupled
encapsulate hydrophobic drug, curcumin into the hydrophobic tris can form co-ordination complex with Fe3+ ions.16 By getting
part of the hydrogel (Figure 4c). Subsequently encapsulated clue from the literature,16 we step-up further to release the
drug was released by breaking the gel by bringing the pH to encapsulated drug by adding Fe3+ solution, as an external
acidic level (Figure 4d). Curcumin encapsulated gel (composite stimuli. In addition to pH, degradation of composite gel was
gel) was found to be reversible with respect to pH and also triggered by adding a solution containing Fe3+ ion. Metal
temperature. Drug release by means of gel degradation is due ion actuated release of curcumin was achieved by adding Fe3+
to the dis-assembly of composite gel in acidic pH and this solution of (1 X 10-3 M) on preformed composite gel and
phenomenon could be visually monitored (Figure 4d). The pH recorded UV absorption of aliquots collected at different
triggered release of curcumin has been identified by adding intervals of time. Gel degradation occurred via slow diffusion
acidic solution (pH=4.0) to composite gel and recorded the UV- of Fe3+ ion into the composite gel. Aliquots collected at various
vis spectra of the supernatant solution periodically. Aliquot interval of time (1 min, 10 min, 20 min, 1 h, 2 h, 3h and 12h)
collected at 0 min did not display any absorbance peak showed absorption maxima at 445 nm, which corresponds to
corresponding to curcumin. As time increases, degradation of the absorption peak of curcumin. From UV-vis measurements,
composite gel started and supernatant solution becomes we could clearly observe the steady release of curcumin from
yellow in color. Aliquot collected at different time intervals the composite gel with respect to time (Figure 4f).
such as 10 min, 20 min and 30 min displays absorbance peak of
Figure 4. (a & b) Photographs of hydrogel and composite gel formed by 4c and its corresponding HRTEM images respectively; (c) photograph of reversible conversion
of gel-to-sol under various pH level and its corresponding HRTEM image; (d) photograph showing the degradation of composite gel under acidic pH and Fe3+ ion and
(e) UV-vis spectra of gelator, 4c dissolved in ethanol, 1X10-5M (black line) and profile of pH as stimuli responsive curcumin release from composite gel at regular
6 | J. Name., 2012, 00, 1-3
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interval of time such as 0 min, 10 min, 20 min and 30 min (red to cyan blue) respectively; (f) UV-vis profile of Fe3+ as stimuli responsive curcumin release from
composite gel at different time intervals such as 1 min, 10 min, 20 min, 1 h, 2 h, 3h and 12h (red to yellow) respectively. For UV-vis measurements, 1 mL of aliquot
was retrieved at different intervals of time and diluted to 2 mL using distilled water. Direction of arrow shows the increase in intensity of curcumin absorption peak
with respect to time.
result suggesting that it possess good tolerance to external
forces.
Figure 5. (a) Proposed binding mode of compound 4c with Fe3+ ions and (b) FT-IR
spectra of xerogel of 4c and its corresponding Fe3+ complex.
Subsequent coordination of Fe3+ with carbonyl and –OH
groups of 4c, as identified using FT-IR spectra, lead to the dis-
assembly of composite gel which directly trigger the drug
release (Figure 5a & 5b). The possible mode of binding of Fe3+
with 4c is given in figure 5a.There are few stimuli responsive
Figure 6. (a) Angular frequency dependence of G′ and G′′ of gel, 4c and composite gel
and (b) strain amplitude dependence of G′ and G′′ of gel, 4c and composite gel.
molecular gels were reported.17 However, only a little is known The rheological property of
regarding the multi stimuli responsive biocompatible hydrogel. independent of strain up to a critical strain level (
This report represents the first example of a pH and metal ion
c, the storage modulus start decay and the material behaves
as stimuli responsive coumarin-tris based hydrogel derived in a non-linear fashion. It is worth to measure G
and G′′ of gel
from a renewable resource.
formed from 4c and its corresponding composite gel in
a
viscoelastic material are
c). Beyond
γ
γ
′
The rigidity and flow behavior of gel formed by 4c were dependence of strain amplitude. Figure 6b shows a strain
investigated. The elastic behavior of a material could be sweep of gel formed from 4c and its composite gel. With
directly identified by measuring the difference between G
′′ 18
Storage moduli (G ) and loss moduli (G′′) of gel formed certain point gradual decrease was observed in a linear fashion
from 4c and composite gel as the function of angular and cross over occurs between G
and G′′, the point at which
frequency (elastic response) is shown in Figure 6. In both the the cross over occurs is considered as critical strain (
c) of a gel.
cases throughout the entire range of frequency sweep, the The γc for gel formed by 4c and its composite gel is 3.88 and
values of G are found to be more than that of G′′. The 3.31% respectively. Below γc (G’>G’’) the self-assembled
difference in these two moduli ∆G (G -G′′) of gel formed by 4c structure is intact and have solid like behavior, and this result
is calculated as 27300 pa, which is comparatively higher than clearly depicts the formation of highly structured material. By
that of corresponding composite gel (∆G = 23440 pa). Thus increasing the strain above
c, disturbs the self-assembled
′ and gradual increase in strain, G
′ and G′′ remains constant and at a
G
.
′
′
γ
′
′
γ
incorporation of drug in the gel network have only little effect network and eventually become fluid-like. The crossover of G
in the rheological behavior (Figure 6a). Visco-elastic behavior and G′′ demonstrate the reversible nature of gel.
of these gels were independent of frequency sweep, and this
′
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Nolte and A. E. Rowan, Chem. Soc. Rev. 2010, 39, 1576–
1599; (d) D. Gonzalez-Rodriguez and A. P. H. J. Schenning,
Chem. Mater. 2011, 23, 310–325; (e) S. I. Stupp and L. C.
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Conclusions
In conclusion, we have designed and synthesised a new class
of coumarin-tris based amphiphiles from renewable resource,
that self-assemble into gels at neutral and basic pH levels, and
vesicles and nanotubes at acidic pH level. Self-assembled
structures were stabilized through the extensive hydrogen
M. B. Hansen and J. C. M. van Hest, Chem. Soc. Rev. 2010, 39
3394–3412; (h) J. D. Tovar, Acc. Chem. Res. 2013, 46, 1527–
,
1537; (i) D. Ivnitski, M. Amit, B. Rubinov, R. Cohen-Luria, N.
Ashkenasy and G. Ashkenasy, Chem. Commun. 2014, 50,
bonding and π-π stacking interactions that are existing in the
gelator. We have demonstrated the reversible sol-gel
switching of coumarin-tris based gel in the presence of pH and
metal ion stimuli. We also depicted the encapsulation of
chemopreventive drug curcumin into the hydrogel, and pH and
Fe3+ triggered encapsulated drug release into the solution also
performed. Rheological studies clearly depicts the stability and
mechanical strength of gel and composite gel. We envision
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that this hydrogel could play
a significant role in
pharmaceutical science, in particular for the development of
new generation of stimuli responsive drug delivery systems for
in vivo formulations.
5
6
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Acknowledgements
This work was financially supported by the Department of
Science and Technology (IFA-CH-04 and #SB/FT/CS-024/2013),
India and Board of Research in Nuclear Science
(#37(1)/20/47/2014), Department of Atomic Energy, India. GJ
acknowledges the support from the Gulf of Mexico Research
Initiative (GoMRI) through the Consortium for the Molecular
Engineering of Dispersant Systems (C-MEDS) subcontract (TUL-
626-11/12).
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7
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4
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