Hierarchical supramolecular hydrogels: Self-assembly by peptides and photo-controlled release: Via host-guest interaction

Article abstract of DOI:10.1039/c7cc07859e

A hierarchical supramolecular hydrogel was self-assembled from a Fmoc-RGDS tetrapeptide and showed photo-controlled release directed by host-guest interaction. Multiple payloads, including vesicles, were successively released from a single peptide hydrogel.

Full text of DOI:10.1039/c7cc07859e

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Hierarchical supramolecular hydrogels:
self-assembly by peptides and photo-controlled
Cite this:DOI: 10.1039/c7cc07859e
release via host–guest interaction†
Received 11th October 2017,
Accepted 26th October 2017
Chih-Wei Chu and Bart Jan Ravoo
*
DOI: 10.1039/c7cc07859e
rsc.li/chemcomm
A hierarchical supramolecular hydrogel was self-assembled from a peptide synthesis (SPPS).21,22,29 Similar to polymeric hydrogels, the
Fmoc-RGDS tetrapeptide and showed photo-controlled release non-covalent interaction of peptide hydrogels also permits the
directed by host–guest interaction. Multiple payloads, including development of multi-stimuli hydrogels. Several stimuli, including
vesicles, were successively released from a single peptide hydrogel. chemical stimulus,30,31 enzyme32,33 and light,34 were reported.
Among them, light as trigger is of particular interest since it
Hydrogels are versatile biomedical materials owing to their allows to target a specific area of the gel with high resolution in
porous structure with high-water content, which have inherent space and time and also, light is a non-invasive stimulus.
biocompatibility and similarity to human tissue.1–3 Supra- Photo-switchable moieties are notably fascinating because they
molecular hydrogels formed either by macromolecules4–7 or small support the establishment of light-responsive peptide hydro-
molecules8–11 have gained increasing interest in recent years. gels. LMWGs comprising azobenzenes35–37 and stilbenes26,38,39
Several research groups have focused on polymeric supramolecular are two important variations of this approach. Azobenzene is
hydrogels based on host–guest interaction and cyclodextrins (CDs) unique since it can be readily photo-isomerized as well as form
are especially useful as excellent host molecules in aqueous photo-responsive inclusion complex with b-CD via host–guest
media.4,5,12–16 Compared to traditional CD-decorated polymers, interaction in water.40 It was also reported that azobenzene
cyclodextrin vesicles (CDVs) offer another possibility to act as containing dipeptides show a photo-induced change of self-
multivalent non-covalent cross-linkers.17 We have reported a poly- assembly in presence of a-CD.41 However, to the best of our
meric supramolecular hydrogel containing CDVs as 3D junctions. knowledge, a light-responsive LMWG based on host–guest
Self-thinning and self-healing properties of the gel were observed interaction is so far unprecedented.
and as a result, an injectable hydrogel was obtained.18 Further-
Herein, we introduce a novel hierarchical supramolecular
more, the non-covalent interaction enables the response of these hydrogel by combination of self-assembly of an amphiphilic
soft materials to various stimuli, for example, light,12–14 peptide and host–guest interaction as additional cross-linking
redox,15,16 metal-ion19 and temperature,20 based on responsive motif to stabilize the gel. Recently, a new type of water soluble
guest molecules.
photoswitch, arylazopyrazole (AAP), has been developed in our
Self-assembly of low molecular weight gelators (LMWGs), on group.42 Its nearly quantitative photo-isomerization combined
the other hand, has also attracted considerable attention in the past with the selective affinity of trans-AAP to b-cyclodextrin facilitate
decade.21 Various LMWGs, such as peptides,22–25 carbohydrates26 better light reversible host–guest systems compared to typical
and synthetic organic compounds,11 have been reported. Aromatic azobenzene.43–45 By introduction of AAP as light-responsive guest
peptides are especially attractive due to their facile synthesis and and CDV as cross-linkers, photo-controlled release of multiple
strong p–p interactions in aqueous solution.23,25 Different sequences payloads from a single peptide hydrogel is achieved.
of amino acids, used as hydrophilic head group, were optimized for
Fmoc-RGDS was chosen as building block for the peptide
various applications.21,27,28 The Fmoc-protecting group is frequently backbone due to its good water solubility and high biocompatibility.
applied as hydrophobic part since it is widely used in solid-phase It has been reported that Fmoc-RGDS can form hydrogel and serve
as cell adhesion motif.27 The p–p interaction between hydrophobic
Fmoc groups induces the self-assembly of the amphiphilic peptide
in water. As the supramolecular polymer grows beyond a certain
length, the entanglement between the self-assembled fibers
reinforces the gel formation (Fig. 1a). Synthesis of Fmoc-RGDS
was carried out by SPPS. To introduce light responsive unit into
Organic Chemistry Institute and Center for Soft Nanoscience,
¨
¨
¨
¨
Westfalische Wilhelms-Universitat Munster, Corrensstrasse 40, Munster 48149,
Germany. E-mail: b.j.ravoo@uni-muenster.de
† Electronic supplementary information (ESI) available: Experimental procedures
and supporting data. See DOI: 10.1039/c7cc07859e
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Ka = 16.9 ꢀ 102 Mꢁ1 was determined, which is comparable to
our previous report.42
The secondary structure of Fmoc-RGDS was studied by
circular dichroism. In accordance with the literature, the self-
assembled Fmoc-RGDS showed a disordered structure and a
low signal at around 200 nm in the circular dichroism
spectrum.27 To further understand the thermal stability of the
self-assembled peptide structure, spectra at different tempera-
ture were recorded. No change was observed in the spectra,
suggesting that the self-assembled peptide is stable up to 45 1C
(Fig. S2a, ESI†). Moreover, insertion of Fmoc-RGDS-AAP into
Fmoc-RGDS has little influence on the self-assembled structure. By
comparison of circular dichroism spectra for 0.01% Fmoc-RGDS
and 0.01% Fmoc-RGDS containing 0.001% Fmoc-RGDS-AAP, a very
similar shape and intensity of the curves was observed (Fig. S2b,
ESI†). These findings show that insertion of Fmoc-RGDS-AAP
did not affect the peptide fibril assembly. However, the slightly
diminished peak at 200 nm suggested that there might be a
secondary p–p interaction between neighboring AAP groups, which
is further explained by microscopic images (see below).
Scanning electronic microscopy (SEM) was exploited to
investigate the surface morphology of the peptide self-assembly.
In this experiment, samples of 2.5% Fmoc-RGDS and 2.5% Fmoc-
RGDS with 0.25% Fmoc-RGDS-AAP were prepared (see ESI† for
sample preparation methods) and multiple areas per sample were
scanned. Fibril structures were found in both cases and entangle-
ment of fibers was also observed, which is consistent with reported
literature.27 In presence of Fmoc-RGDS-AAP, SEM showed more
cross-linking fibers (Fig. 3) compared to only Fmoc-RGDS (Fig. S3,
ESI†). A reasonable explanation for this observation might be the
formation of additional p–p interactions between AAPs protruding
from the peptide fibrils.
Fig. 1 (a) Schematic representation of the self-assembly of Fmoc-RGDS
in water and (b) structure of AAP-functionalized peptide Fmoc-RGDS-AAP
obtained via CuAAC and SPPS.
peptide backbone, Fmoc-L-Ser-TEG-AAP was synthesized via
copper-catalyzed azide–alkyne cycloaddition (CuAAC) and used
as first amino acid in SPPS. Fmoc-RGDS-AAP was obtained after
stepwise elongation. Details of the synthesis and analysis of the
peptide are provided as ESI.†
To investigate the photophysical properties of Fmoc-RGDS-
AAP, UV/vis spectroscopy was used. AAP is a light-responsive
motif and undergoes isomerization upon UV (365 nm) and
visible (520 nm) irradiation. Indeed, Fmoc-RGDS-AAP displayed
a similar spectrum as our first report on AAP,42 i.e. a p - p*
band at 334 nm and an n - p* band around 430 nm. Under UV
irradiation (365 nm), the p - p* absorbance is diminished
whereas the n - p* band is increased. This observation reveals
that the trans- to cis-isomerization is complete after 15 min
irradiation. After irradiation with visible light (520 nm) for
15 min, the original spectrum is obtained, indicating near
complete cis- to trans-isomerization. The slightly higher absorbance
at 334 nm compared to the original spectrum may be due to the
presence of cis-isomer as trace amount in the peptide as prepared.
Upon UV and vis irradiation, the absorbance of the Fmoc group
between 250–300 nm remains almost constant (Fig. 2a). The photo-
induced switching of Fmoc-RGDS-AAP is fully reversible for at least
three cycles (Fig. 2b).
Next, a light responsive hierarchical supramolecular hydro-
gel was developed by combination of Fmoc-RGDS, Fmoc-RGDS-
AAP and CDV. The molecular structure, size and preparation of
CDV are provided in the ESI† (Fig. S6). The average size of CDV
obtained by DLS is ca. 140 nm. Hydrogels were prepared by
dissolving several milligrams of peptides in ddH2O, followed by
gently heating at 45 1C for 5 min. After gelation overnight, all
gels passed the ‘‘reverse vial’’ test. Various compositions were
tested and versatile light-responsive hydrogels were achieved in
presence of 2.5% Fmoc-RGDS (38 mM), 0.25% Fmoc-RGDS-AAP
(2.3 mM) and 50 mM CDV (Fig. 4). The hydrogels were formed
not only by entanglement of self-assembled supramolecular
Another appealing feature of AAP is that it provides not only
superior photoswitching behavior but moreover trans-AAP has
strong affinity to b-CD, which broadens the application in
cyclodextrin-based supramolecular systems. Host–guest inter-
action between Fmoc-RGDS-AAP and b-CD was investigated by
ITC (Fig. S1, ESI†). A 1 : 1 stoichiometry and a binding constant
Fig. 2 (a) UV/vis spectra of trans- and cis-isomers of Fmoc-RGDS-AAP
and (b) three cycles of fully reversible photo-isomerization of Fmoc-
RGDS-AAP.
Fig. 3 SEM images of 2.5 wt% Fmoc-RGDS in presence of 0.25 wt%
Fmoc-RGDS-AAP at (a) 10kꢀ and (b) 25kꢀ magnification.
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loss modulus (G00) was found in hydrogels containing of 200 mM
CDV, whereas the lowest was observed for hydrogels containing
only 25 mM CDV. This observation shows that a higher concen-
tration of CDV leads to a higher degree of cross-linking so that a
more rigid supramolecular material is obtained. Since the hydrogel
is based on supramolecular polymers, the response to shear is
interesting to examine (Fig. S4b, ESI†). Initially, a lower shear rate
(g = 0.5 sꢁ1) was applied and constant viscosity for all hydrogels was
_
observed. Then, a high shear rate (g = 500 sꢁ1) resulted a decline in
_
viscosity, indicating disruption of the supramolecular structure
under shear. Finally, a rapid recovery of viscosity was received after
lowering shear rate back to 0.5 sꢁ1. Thus, all hydrogels showed
shear-thinning behaviour (thixotropy). Furthermore, the change of
viscoelastic properties under irradiation were studied. A frequency
sweep measurement of samples comprising 2.5 wt% Fmoc-RGDS,
0.25 wt% Fmoc-RGDS-AAP and 50 mM CDV was recorded. In this
case, rheology of the hydrogel showed a large decrease of both
storage (G0) and loss modulus (G00) after UV irradiation. Upon
visible light irradiation, although the plateau modulus (G0) did
not recover quite as high as before, over one order increase was
achieved (Fig. 5b). These rheology measurements clearly show that
the hierarchical peptide gel is stabilized by host–guest interaction
of cyclodextrin in the CDV and AAP on the peptide and that
alternating photo-isomerization of trans-AAP to cis-AAP induces a
reversible softening and stiffening of the gel.
To investigate this supramolecular hydrogel as photo-
controlled release system, two fluorescent dyes with different
molecular weight (FITC-isomer I, MW = 389 g molꢁ1 and FITC-
Dex4000, MW = 4000 g molꢁ1) were encapsulated inside the
hydrogel. 200 mL of hydrogel samples were prepared one day
before (see ESI† for preparation details) at the bottom of
cuvettes. And additional 800 mL of ddH2O was added carefully,
minimizing the disruption of hydrogel interface. Release of
fluorescent dye was monitored by fluorescence spectroscopy
with and without UV irradiation (Fig. 6). The percentage of
release was calculated by dividing the fluorescence intensity of
fully dissolved hydrogels obtained by heating and gently stirring.
Significant releases of both dyes were detected under UV irradia-
tion for 4 h. FITC-Dex4000 showed a slower and less extensive
release, which means that it was trapped longer and tighter
inside the gels due to its larger size than FITC-Isomer I. (Fig. 6a,
red and blue lines). Control experiments were also performed by
monitoring diffusion of fluorescent from hydrogel to aqueous
solution in the dark. No significant release was observed within
4 h, but depending on the molecular weight of dyes, 20% and
55% of release were observed after 20 h (Fig. S5, ESI†).
Fig. 4 Reversible photoswitching of a supramolecular hydrogel composed
of 2.5 wt% Fmoc-RGDS, 0.25 wt% Fmoc-RGDS-AAP and 50 mM of CDV
(scale bar: 1 cm).
polymers but additionally by host–guest interaction between AAP
and CDV. After irradiation with UV-light, the self-supporting
hydrogel was weakened due to loss of host–guest interaction,
resulting from isomerization of trans-AAP to cis-AAP. Since cis-
AAP does not bind to b-CD, the non-covalent interaction between
supramolecular fiber and CDV is strongly reduced. As a result, a
very soft, non-self-supporting gel was obtained. Reversible stif-
fening of the hydrogel was demonstrated by storing the very soft
gel either in dark or under visible light. After one day, the sample
kept in dark remained as very soft gel while the sample that was
exposed to light recovered as a self-supporting hydrogel. The
stiffening of the hydrogel is due to reconstruction of host–guest
interaction between trans-AAP and CDV. Since the half-life time
of isomerization from cis- to trans-AAP is approximately four
days,42 the sample kept in dark cannot recover as a hydrogel
within one day. This observation supports our claim that the
light responsive hydrogel is stabilized via host–guest interaction.
Rheological measurements revealed the visco-elastic proper-
ties of the supramolecular hydrogels. Two different concentra-
tions of Fmoc-RGDS were compared in oscillatory rheological
measurements. In Fig. S4a (ESI†), 5% Fmoc-RGDS showed
approximately two orders of magnitude higher storage modu-
lus G0 than 2.5% Fmoc-RGDS. These findings suggested that
stiffer gels can be obtained with a growing number of entangle-
ments of the peptide fibers. Similar observations were made if the
concentration of CDV was increased (Fig. 5a). Hydrogels containing
2.5 wt% Fmoc-RGDS, 0.25 wt% Fmoc-RGDS-AAP and increasing
concentration of CDV were compared. The highest storage (G0) and
Another appealing feature of this responsive soft material is
the photo-controlled release of CDV from the supramolecular
peptide hydrogel. In order to examine CDV release from hydro-
gels, hydrophobic NBD-cholesterol was embedded in the
membrane of CDV. Otherwise, the same experimental setup
was employed as described above (Fig. 6b). Compared to the
release of FITC-I and FITC-Dex4000, the release of CDV is
considerably slower. A significant release of CDV is only
observed after 2 h of irradiation (Fig. 6a, black line). This result
is consistent with our expectation that the rate of release from
Fig. 5 (a) Frequency sweep of oscillatory rheological measurements
performed at 0.5% strain for increasing concentration of CDV. (b) Photo-
switching experiment performed in presence of 2.5 wt% Fmoc-RGDS,
0.25 wt% Fmoc-RGDS-AAP and 50 mM CDV.
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We are grateful for the financial support from EC H2020 –
Marie Skłodowska-Curie Actions – Innovative Training Network,
Multi-App (project number: 642793). We thank PD Dr Cramer-
¨
¨
Kellers and Mrs Probsting for advice on rheology. Markus Borner
is acknowledged for SEM measurements and Luca Cochet and
Benedikt Nowak are acknowledged for peptide synthesis.
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Conflicts of interest
38 S. Matsumoto, S. Yamaguchi, S. Ueno, H. Komatsu, M. Ikeda,
K. Ishizuka, Y. Iko, K. V. Tabata, H. Aoki, S. Ito, H. Noji and
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There are no conflicts to declare.
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