Light-triggered syneresis of a water insoluble peptide-hydrogel effectively removes small molecule waste contaminants

Article abstract of DOI:10.1039/c9cc09225k

A short peptide based hydrogel exhibits aqueous insolubility, thixotropy and efficient light induced syneresis. Upon irradiation with UV light, the hydrogel shrinks and expells ～50% of the solvent. Syneresis is caused by light-triggered trans-cis isomerisation of an azobenzene moiety in the peptide derivative. This expulsion of solvent can be effectively exploited in the removal of low molecular weight contaminants in water.

Full text of DOI:10.1039/c9cc09225k

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Light-triggered syneresis of a water insoluble peptide-hydrogel
effectively removes small molecule waste contaminants†
Basab Kanti Das,a ‡ Bapan Pramanik,a ‡ Sumit Chowdhuri,a Oren A. Schermanb and Debapratim
Received 00th January 20xx,
Accepted 00th January 20xx
Das*a
DOI: 10.1039/x0xx00000x
A short peptide based hydrogel exhibits aqueous insolubility, developed to design such gelators only with the help of more
thixotropy and efficient light induced syneresis. Upon irradiation examples and analyses.
with UV light, the hydrogel shrinks and expells ~ 50% of the solvent.
Syneresis is caused by light-triggered trans-cis isomerisation of an
azobenzene moiety in the peptide derivative. This expulsion of
solvent can be effectively exploited in the removal of low molecular
weight contaminants in water.
Supramolecular hydrogels made of small peptides have been
attracting interest for several decades on account of potential
applications in areas such as drug delivery, materials science,
sensing and nano-technology to name a few.1-4 These systems
have been rendered stimuli responsive through the
introduction of pH, heat, metal ions and light.5-8 Various
supramolecular hydrogels have also been reported with unique
properties including aqueous insolubility, stretchability,
injectability, unusual aging and strength.9-13 However, reports
regarding syneresis or macroscopic volume phase transitions in
response to stimuli are rare for supramolecular gels. Syneresis hydrogel formed by the solution (H-gel) and syneresis (S-gel) upon irradiation with
is the expulsion of liquid from a gel; a common example is the mentioned in (B).
discharge of blood serum during the clotting process.14
Scheme 1. A) Chemical structure of Azo-KC. B) Photographs of the Azo-KC solution,
UV light. C) Possible molecular arrangements corresponding to different states as
In this regard, we have recently reported a peptide 9, 21 capable
of forming hydrogels, which remain insoluble in bulk water and
exhibited an unusual property as exchange of solvent and solute
to and from these hydrogels is highly restricted. Dimerisation of
the gelator was found to be critical for this unique property.
Exploring the possibility of creating new hydrogelators with
similar effect, we have prepared an azobenzene-functionalised
short peptide, Azo-KC (Scheme 1), which efficiently formed
hydrogels under basic conditions. In addition to insolubility, the
hydrogel also displayed light-triggered syneresis. Additionally,
the hydrogel found to be responsive toward disulphide-bond
breakers and thixotropic property. The combination of all these
properties like, dual responsiveness, syneresis, insolubility and
injectability make the hydrogel distinctive amongst all such
supramolecular hydrogel reported so far. Here, we report the
hydrogelation by Azo-KC, its aqueous insolubility, light-driven
syneresis and its ability to effectively remove contaminants
from water.
Relatively few examples of supramolecular gels exhibiting
syneresis have been reported to date.15-18 These hydrogels
exhibited contraction and expulsion of water in response to
various stimuli such as, pH,19 metal ions,18 light,17 mechanical
force16 or heat17. Such a property can lead to various important
applications in the field of material science and bio-medical
field. While an effort was made by Adams and co-workers to
propose a rational behind such behaviour in a supramolecular
gel,20 it is still not clear why and when such property could be
expected. Probably, all the reported hydrogels are
serendipitous observations and a proper model could be
a Department of Chemistry, Indian Institute of Technology Guwahati, Assam
781039, India. Email: ddas@iitg.ac.in
b Melville Laboratory for Polymer Synthesis, Department of Chemistry, University
of Cambridge, Cambridge CB2 1EW, United Kingdom.
‡Authors contributted equally.
† Electronic Supplementary Information (ESI) available: [Experimental details,
characterization data and other supporting figures]. See DOI: 10.1039/x0xx00000x
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An aqueous solution of Azo-KC forms a self-supporting hydrogel these media after 7 d are shown in Fig. 1D. The hydrogel
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(H-gel, Scheme 1) upon treatment with base (NaOH). The remained insoluble even after one yearDoOfI: i1n0c.1u0b3a9/tCio9nCCin09b22u5lKk
minimum gelation concentration (MGC) and gel to sol transition water.
temperature at MGC were found to be 1.15 wt% (16.2 mM of The self-assembly of Azo-KC is assisted by the disulphide bridge
Azo-KC) and 57 °C, respectively. Transmission electron between two peptides. After 2 h of incubation under basic
microscopic (TEM) analysis shows formation of a network of conditions, only the mass corresponding to the Azo-KC dimer
thin fibers (2-5 nm
× ~100 nm; Fig. 1A). Rheological was found. Time-dependent high pressure liquid
measurements confirmed a gelled state of the material with a chromatographic (HPLC) analyses of a diluted solution of Azo-
higher storage modulus (G’) compared to its loss modulus (G”) KC shown in Fig. 2A revealed that the dimerisation is complete
in both amplitude and frequency sweeps (Fig. 1B and S1).22 within 1.5 h. The highly basic medium in this case presumably
While
performing
the
strain-dependent
rheology facilitates oxidation of the Cysteine thiols.21 To understand the
measurement, the hydrogel showed a yield strain and situation in the gel state, a portion of the H-gel was dissolved in
transforms into a quasi-liquid indicating possible thixotropic DMSO and further diluted with water and analysed using
(injectable) behaviour.23
A time-dependent strain sweep analytical HPLC. More than 99% of the molecules were found to
experiment was performed by alternating the applied strains at be in the dimer form (Fig. S5). To demonstrate the importance
a fixed angular frequency. At a higher strain ( = 1000%), the of the disulphide linkage towards gelation, the hydrogels were
viscoelastic properties of the material were lost, but were placed into aqueous solutions containing various disulphide
regained almost completely in every successive steps when a bond-breaking agents. In all cases the hydrogel dissolved within
lower strain ( = 0.1%, Fig. 1C) was applied. This step-strain minutes (Fig. S3).
measurement was cycled four times with excellent recovery,
demonstrating thixotropic behaviour by the hydrogel. The
hydrogel could easily be injected through a syringe immediately
recovering as a gel after shear (Fig. S2).
Fig. 2 A) Time dependent chromatographic analysis of a 0.01 wt% aqueous
solution of Azo-KC showing the formation of disulfide linked dimer. B) 1H NMR
Fig. 1 A)
spectra of Azo-KC dimer in DMSO-d6 with varying amounts of H2O showing the
down-field shifts of the amine NHs and up-field movements of the aromatic
protons. All measurements were carried out at room temperature.
FESEM
1
A H NMR was carried out on solutions of Azo-KC in DMSO-d6
after 12 h with varying amounts of water. This revealed that
both hydrogen bonding and  stacking interactions become
prominent in the aggregated state.24 With an increase in water
content, the amide-NHs showed down-field shifts while the
aromatic protons moved upfield (Fig. 2B). Powder XRD analyses
of the as synthesised Azo-KC and xerogel obtained from the H-
gel confirmed  stacking between the “Azo” groups
(interplaner distance of 4.23 Å) in the self-assembled state (Fig.
S6). Fourier-transform infrared (FTIR) spectra of the as
synthesised peptide showed two peaks at 1683 and 1652 cm-1,
which were shifted to 1673 and 1639 cm-1, respectively in the
H-gel (Fig. S7). The observed shifts and peak positions in the H-
gel indicate possible hydrogen bonding between the
neighbouring peptide molecules.25
The gelator, Azo-KC, was designed to embed a light sensitive
moiety into a hydrogel by incorporating an “Azo” group, which
can undergo trans to cis isomerisation upon irradiation with UV
light.17, 26 When irradiated with light ( = 365 nm), the H-gel
displayed shrinkage, expelling water from the hydrogel (Fig.
3A). This unexpected observation lead us to perform a
systematic evaluation of hydrogel syneresis. Quantitative
analysis showed that the hydrogel can shrink up to maximum of
50% of its original volume following irradiation with UV light
(Fig. 3A). It is clear from the plot in Fig. 3A that the shrinkage
process is complete within 90 min. HPLC (Fig. S5) and ESI-MS
image of the H-gel. B) Changes in storage and loss moduli as a function of shear
strain for H- and S-gels. C) The rheological properties of 1.15 wt% H-gel when
alternate step strain switched from 1% to 200%. D) % dissolution of the H-gel in
buffers of different pH (1-13) after 7 days of incubation at room temperature.
It is important to note that, unlike cross-linked polymeric
hydrogels, the formation of supramolecular hydrogel networks
are entirely governed by non-covalent weak forces. Thus, when
dispersed in bulk water, these networks disassemble and the
individual gelator molecules become solubilised which results in
an overall gain in entropy of the system. However, in line with
our previous report,9 the hydrogel formed by Azo-KC was also
found to be insoluble in water. A small portion of the hydrogel
was placed in bulk water or buffers at different pH values (1-13)
and shaken at a constant speed. The bulk medium was analysed
over time using UV-Vis spectroscopy to quantify the dissolution
(Fig. S3-4). In all media, an initial dissolution (up to 11%) was
observed, corresponding to loosely-bound gelator molecules at
the surface of the hydrogel. However, in all cases, after 24 h no
further dissolution was observed. The extent of dissolution in
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analyses of the shrunken gel (S-gel, Scheme 1, Fig. 3A) confirm suggest a significant alteration takes place in the self-assembly
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DOI: 10.1039/C9CC09225K
that the constituent molecule of S-gel remains the disulphide of the gelator molecules upon irradiation with UV light.
linked dimer of Azo-KC. The HPLC analyses shows ~ 9:1 The light induced “trans” to “cis” structural isomerisation of
distribution of cis and trans isomers of the dimer in the S-gel Azo-KC changes the self-assembly state. The UV-Vis absorption
sample.
spectra of a solution of Azo-KC dimer was recorded at different
To check reversibility of the syneresis process, the shrunken gel time intervals while irradiating the solution with light (365 nm).
was further irradiated with light of 420 nm as well as kept in Before irradiation, the molecule showed an absorption maxima
direct sunlight for several hours with occasional shaking. at 342 nm (Fig. 3C), which corresponds to the  transition of
However, no change in the appearance was observed, which the trans-azobenzene unit.27 As the sample was irradiated with
confirmed irreversibility of the process. Water expulsion was UV light, a hypsochromic shift was observed in the absorption
also monitored by keeping the H-gel at room temperature for maxima. After 90 min of irradiation, the absorption maxima was
several days. However, no syneresis was observed in this case. 321 nm and the molar extinction coefficient decreased linearly
Finally, the hydrogel was heated at 45 °C and only a small for the respective absorption maxima. Additionally, a new
portion (~ 15%) of the total water was expelled from the broad peak at 445 nm appeared in the irradiated sample
hydrogel after 2 h of heating. Thus, though heating can cause increasing with irradiation time. This new peak is a signature of
syneresis in this system, UV light irradiation was found to be a the n–* transition of cis-azobenzene.27
more effective stimulus.
Circular dichroism (CD) of the Azo-KC hydrogel revealed a
bisignate signal in the azobenzene absorption region with a
positive to negative crossover point at 324 nm (Fig. 3D).
Additionally, a positive peak at 198 nm and a negative Cotton
effect at 222 nm were also observed indicating -sheet like
arrangement of the peptide. The strong exciton-coupled cotton
effect at the bisgnate peak is a result of supramolecular chirality
in the aggregated state.17 While the secondary conformation of
the peptide remained similar for the S-gel, the 250-400 nm
region remained CD silent. PXRD of the xerogel formed from the
S-gel showed two  stacking interactions (4.16 and 3.79 Å)
compared to only a single peak for the H-gel (Fig. S6). However,
the hydrogen bonding interactions are still observed in the IR
spectra with two prominent bands at 1677 and 1637 cm-1
supporting a -sheet like arrangement similar to that of the H-
gel (Fig. S7).
Fig. 3 A) % of water expelled from the H-gel during syneresis as a function of
duration of the UV-irradiation process. Inset: images of the vials after 2 h of
irradiation as in (A) showing the expelled water. B) FESEM image of the S-gel. C)
UV-Visible spectra of a 0.01 wt% 12 h matured aqueous solution of Azo-KC (in
presence of NaOH) during UV-irradiation. D) CD spectra of a 0.01 wt% 12 h
matured aqueous solution of Azo-KC (in presence of NaOH) before and after 2 h
of UV-irradiation. All measurements were carried out at room temperature.
The syneresis observed in this system is an interesting
phenomenon, thus gaining mechanistic insight into the system
is important for enhanced understanding. The S-gel was
analysed using electron microscopy as well as rheology.
Interestingly, the S-gel consists of ~ 1 m long rod-like
structures (Fig. 3B). This morphology clearly differs from the
original network of thin fibers observed for the H-gel. The
sample after 30 min of exposure to UV light shows a
combination of both fibers and rods, which reflects an
intermediate state of the morphogeneses process (Fig. S8). For
the S-gel, both storage and loss moduli were found to be much
higher than that of the H-gel (Fig. 1B and S1). These results
Fig. 4 A) Photographs showing MO removal from H-gel upon UV light induced
syneresis. B) UV-Vis spectra of MO and the expelled water from (A). C) Maximum
initial dye concentrations for different dyes, which can be completely removed
through syneresis of the hydrogel. All measurements were carried out at room
temperature.
It is important to note that the transformation in morphology
occurs while the system is in an aggregated gel state where the
dynamics of the constituent molecules are highly restricted.21
Crystal to crystal transformation due to photo-induced trans-cis
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isomerisation of azobenzene derivatives has been reported in
literature.28 However, such gel-state morphogenesis are not
commonly observed phenomenon.29 It is clear that during the
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trans-cis isomerisation, the Azo-KC dimer undergoes
a
significant change in molecular arrangement, which is shown in
Scheme 1. Presumably the new arrangement in the S-gel
requires less water to stabilise the aggregated structure. Thus,
the system expels excess water present in the system.
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be used to entrap toxic small molecules, including dyes, in the
S-gel structure. Six different model dyes were tested. H-gels (at
MGC) were prepared in 20 M solutions of the model dyes. The
H-gels were then subjected to syneresis by irradiation with UV
light (365 nm) for 2 h. The expelled water was then analysed
using UV-Vis spectroscopy to quantify the concentration of dye
expelled. Importantly, the expelled water was found to be free
of dye in all cases. Fig. 4A-B show the results for methyl orange
(MO) while the results of other dyes are reported in the ESI (Fig.
S9-10). Keeping the gelator concentration fixed at MGC,
different initial concentrations of the dyes were then evaluated.
For MO, the syneresis process could remove the dye completely
at or below 120 M initial dye concentration as analysed by UV-
Visible spectra (Fig. 4B) and analytical HPLC (Fig. S10). Above
this concentration, a small amount of MO was found in the
expelled water. For other dyes, the results were similar and are
shown in Fig. 4C.
In summary, we have presented a small peptide based hydrogel,
which displayed insolubility in water as well as buffers of
different pH (pH 1-13). The hydrogel was found to be thixotropic
in nature and exhibited efficient syneresis upon irradiation with
UV light. The syneresis is stimulated through light-induced
trans-cis isomerisation of the gelator molecule, which
rearranged within the gelled state and expel the excess
unwanted water. The syneresis process was successfully utilised
to efficiently remove model dyes from water.
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Conflicts of interest
There are no conflicts to declare.
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DD and OAS acknowledge financial support from UKIERI
(DST/INT/UK/P-119/2016 and DST UKIERI 2016-17-011
respectively).
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Notes and references
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DOI: 10.1039/C9CC09225K
Syneresis: A water insoluble hydrogel that expels 50% of the
water upon irradiation with UV-light.
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