Construction of self-assembled nanostructure-based tetraphenylethylene dipeptides: Supramolecular nanobelts as biomimetic hydrogels for cell adhesion and proliferation

Article abstract of DOI:10.1039/d0tb01147a

Supramolecular fluorescent materials with aggregation-induced emission (AIE) characteristics have promising applications as fluorescent probes for bio and chemosensors. In this study, a versatile low molecular weight tetraphenylethylene dipeptide hydrogelator (TPE-YY) with efficient AIE characteristics was developed, which was able to form hydrogels in a broad pH range from 3.7 to 10.2. The self-assembly of this hydrogel is completely pH-dependent, with significant structural transitions from high to low pH. At a relatively high pH value (10.2), a weak transparent hydrogel with an entangled network of nanofibers was obtained, while upon neutralization (pH 7.2) with dilute HCl, a stable semi-transparent gel with twisted nanobelts was formed. When the pH of the hydrogel was reduced to below 5.7, a turbid viscous gel and precipitation appeared with the thickening of the nanobelts. Surprisingly, the hydrogel resulting from the glucono-δ-lactone triggered gel showed only nanofibers independent of pH. The nature of the hydrogels and the nanostructures at different pH values were thoroughly examined and discussed via oscillatory rheology, electron microscopy and various spectroscopic techniques {1HNMR, 2D-NMR, Fourier transform infrared (FTIR) and Circular dichroism (CD)}. Interestingly, the self-assembled hydrogel exhibited excellent biocompatibility over 95% using hydrogel leachables on two different cell lines, 3A6 (human MSCs) and L929 (mouse fibroblast cells). Moreover, the self-assembled nanobelts formed at neutral pH showed excellent cell adhesion and proliferation of 3A6 cells, whereas the nanofibers showed poor cell adhesion and proliferation. Overall, we demonstrate the importance of chemical design for the formation of self-assembled nanobelts and supramolecular hydrogels at physiological pH with selective cell adhesion and proliferation, presenting a promising applications in tissue engineering and regenerative medicine. This journal is

Full text of DOI:10.1039/d0tb01147a

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Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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J Po lue ran sael od fo Mn aot te rai ad l jsu Cs ht emm ai rs gt ri ny sB
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DOI: 10.1039/D0TB01147A
ARTICLE
Construction
of
Self-Assembled
Nanostructures
based
Tetraphenylethylene Dipeptides: Supramolecular Nanobelts as
Biomimetic Hydrogel for Cell Adhesion and Proliferation
Received 00th January 20xx,
Accepted 00th January 20xx
Satish Kumar Talloj, Mohiuddin Mohammed and Hsin-Chieh Lin*
DOI: 10.1039/x0xx00000x
Supramolecular fluorescent materials with aggregation induced emission (AIE) characteristics have gained promising
applications as fluorescent probes for bio and chemosensors. In this study, a versatile low molecular weight
tetraphenylethylene dipeptide hydrogelator (TPE-YY) with efficient AIE characteristics was developed, which was able to
form hydrogel under a broad pH range from 3.7 to 10.2. The self-assembly of these hydrogels is completely pH dependent
with significant structural transitions from high to low pH. At relatively high pH (10.2), a weak transparent hydrogel with
entangled network of nanofibers is resulted, while upon neutralization (pH=7.2) with dilute HCl, a stable semi transparent
gel with twisted nanobelts is formed. As the pH of the hydrogels is reduced to below 5.7, a turbid viscous gel and
precipitation occurred with thickening of the nanobelts. Surprisingly, the hydrogel resulting from Glucono--lactone
triggered gel showed only nanofibers which is independent of pH. The nature of the hydrogels and the nanostructures at
different pH were throughly examined and discussed by using oscillatory rheology, electron microscopy and various
1
2
spectroscopic techniques { H NMR, D-NMR, Fourier transform infrared (FTIR) and Circular Dichroism (CD)}. Interestingly,
the self-assembled hydrogel exhibited excellent biocompatibility over 95% using hydrogel leachbles on two different cell
lines 3A6 (Human MSCs) and L929 (mouse fibroblast). Moreover, the self-assembled nanobelts forming at neutral pH
showed an excellent cell adhesion and proliferation of 3A6 cells, whereas the nanofibers showed poor cell adhesion and
proliferation. Overall, we demonstrate the importance of chemical design for the formation of self-assembled nanobelts
and supramolecular hydrogel at physiological pH with selective cell adhesion and proliferation representing promising
applications in tissue engineering and regenerative medicine.
demonstrated the self-assembling capabilities of short peptides
in aqueous environment could be improved by capping with
aromatic residues such as fluorenylmethoxycarbonyl (Fmoc),
Introduction
naphthalene (Nap), naphthalene diimide (NDI), pyrene (Py),
etc., can facilitate the formation of hydrogel through and
hydrophobic interactions. Similarly, the self-assembly of
aromatic diphenylalanine (FF) derivatives generate nanofibers,
tubes, and nanoparticles by slightly changing the synthetic
Self-assembly of low molecular weight hydrogelators open up
excellent opportunity to control chemical functionality and
morphology and thus biological activity. Polypeptides or
proteins will tend to self-assemble in water into various
-
1-3
4
5
supramolecular nanostructures such as micelles, nanofibers,
19,20
6
7
8
conditions.
sequence or bioactive peptides that spontaneously organize
into -sheet structures in aqueous solution at physiological pH
is an essential condition for many biomaterial applications.
Finally, by choosing the suitable peptide
nanotubes, nanobelts and nano-ribbons under specific
conditions. Firstly, two or more amino acids connected by
amide linkages were synthesized by adopting straightforward
synthetic protocol such as solid phase peptide synthesizer to

21,22
9
Moreover, there is a growing body of evidence suggesting that
there is a relationship between peptide based nanomaterials
form short peptides that will serve as building blocks. Secondly,
the peptide self-assembly can be engineered with variety of
molecular components at N-terminals for the creation of
23
and cell adhesion and proliferation. For instance, Zhang et al.
developed an ionic self-complementary peptide
15-18 (AEAEAKAKAEAEAKAK), EAK16 based hydrogels for cell
and Lin et al,
10
supramolecular nanostructures with different morphologies.
11,12
13,14
For example, Ulijn et al,
Adams et al,
24
adhesion and growth. The interaction between the positively
charged lysine (K) and negatively charged glutamic acid (E) side
chains in EAK16 played an important role in its self-assembly. In
a recent study, the self-assembled hydrogels of glycosylated
peptides shown great potential for cell attachment and
Department of Materials Science and Engineering, National Chiao Tung
University, Hsinchu 30010, Taiwan. Email: hclin45@nctu.edu.tw
Electronic Supplementary Information (ESI) available: Synthetic Experimental
details, 1H-NMR, C-NMR, additional figures and schemes (synthetic schemes for
peptide preparation). See DOI: 10.1039/x0xx00000x
2
5,26
growth.
In a similar study, we demonstrated a glucosamine
13
capped PFB-F self-assembled into supramolecular nanotubes to
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promote wound healing for human mesenchymal stem cells.27
Therefore, we believe that by molecular engineering at N-
terminal of the peptides and by pH switching, one could
precisely control the nanostructures for diverse materials
applications under specific parameters.
View Article Online
DOI: 10.1039/D0TB01147A
Currently, supramolecular fluorescent materials (SFM) with
aggregation induced emission characteristics have been
considered as promising materials for bio probes,
chemo/biosensors in biomedical applications.28,29 In this study,
we chose SFM as tetraphenylethylene (TPE), because of its
facile synthesis and can be engineered with any bioactive
peptides. This unique propeller shaped TPE material is strongly
emissive in the aggregated state owing to the well-known
property of aggregation induced emission (AIE) due to
restricted intramolecular motion (RIM).30 Many researchers
have been studied the functions of TPE containing organic Scheme. 1 Schematic representation of TPE-YY hydrogel A) for the formation of tailored
materials for bio imaging. However, there are very few reports nanostructures with pH; B) twisted nanobelts for selective cell adhesion response and C)
of TPE with short peptides as hydrogelators. We reported useful as a bio-imaging probe.
recently an ultra-short dipeptide TPE-GG tends to be a
hydrogelator under basic media (pH=10.5)31 and while the TPE- representing TPE-YY as a potentially excellent scaffold for stem
single amino acid conjugates such as serine (S) and aspartic acid cell-based therapies (Scheme. 1). Overall, the current work may
(
D) undergoes self-assembly to form hydrogel in acidic provide new insights for the design of chemical structure, pH
3
2
environment (pH=6.1)
with nanosheets morphology. and morphology relationships to develop a new generation of
Therefore, these materials failed to form hydrogels at neutral biomaterials for tissue engineering applications.
pH conditions which is considered as main drawback for
biomaterials. For tissue engineering applications, materials that
could form a self-assembled hydrogel at pH 7.2 to 7.4 is a
Results and Discussions
prerequisite; therefore, in the present study we engineer the
hydrophobicity of the dipeptides by incorporating with
aromatic amino acids such as phenylalanine (F) and tyrosine (Y).
However, the conjugates of FF with TPE derivatives tends to be
too hydrophobic (Hydropathy index* for F=2.8), insoluble in any
aqueous conditions and thus difficult to form a hydrogel.31,33
The TPE-YY material used in this study is prepared by standard
solid phase peptide synthetic protocol (Scheme S1 and S2). The
study begins by testing the hydrogelation propensity in
deionized water at various pH and concentrations. The 2.0 wt%
of the hydrogelator was dissolved in water by adding 1M NaOH
solution and then carefully adjusted the pH using dilute HCl
solution to form a supramolecular hydrogel. At pH 12.0, TPE-YY
was completely in ionized state and the self-assembly is not
much favored due to electrostatic repulsions and hence no
hydrogel was observed. When the pH was reduced to 10.2 by
adding dilute HCl a weak transparent hydrogel was resulted
with very fast gelation kinetics . By the addition of excess HCl
with uniform stirring a semi transparent hydrogel at neutral pH
[
*Hydropathy index is the term used to represent the
hydrophobic/hydrophilic properties for the amino-acid side
34
chains; higher the value, greater is its hydrophobicity] . While,
tyrosine having an extra hydroxyl group on phenyl ring has a
34
negative hydropathy index of -1.3 , govern the self-assembly
by additional hydrogen bonding interactions and hence it is
more water soluble and the gelation abilities of tyrosine
derivatives can be controlled by enzyme triggered
dephosphorylation mechanism, thereby describing it as a better
(
7.2) was obtained. As the concentration of the acidic nature
increases further, opaque to turbid gels with pH 5.5 to 3.7 are
formed (Fig. 1A). Beyond 3.7, a phase separation with water and
precipitate was resulted. We confirmed the gelation by
inverting the vial for 3 to 5 minutes, if no gravitational flow of
water is observed then the material is considered as gel. These
hydrogel materials are expected to show bright blue
fluorescence (Fig. S1A) due to restricted motion of TPE group in
the aggregated state when exposed to hand held UV light of
λ=365 nm. In a highly dissolved state (pH=12.0), TPE-YY
exhibited no or weak fluorescence, however, with the addition
of dilute acid, the solution becomes viscous and RIM process
becomes active due to slower rate of intramolecular rotations
of TPE. Notably, the fluorescence was intensified at pH 4.0 due
to the formation of insoluble aggregates. We then tested
minimum gelation concentration (0.5, 0.75, 1.5 and 2.0, wt%)
3
5,36
hydrogelator.
Based on the above strategies, herein we prepared a low
molecular weight homo dipeptide such as tyrosine conjugated
with a TPE group that was able to form a self-supportive
hydrogel with broad range of pH (3.7 to 10.2). To the best of our
knowledge this was known to be the first of its kind with TPE
group as capping agent based short peptides. Interestingly, we
also demonstrated how the nanostructures were manipulated
or controlled while tuning the pH with dilute HCl. The transition
of nanofiber to flat nanobelts and then to twisted nanobelts
were clearly shown by decreasing the pH of the hydrogel. These
distinct morphologies with pH variations have special interests
in many biotechnological applications. Interestingly, the
nanobelts at physiological pH were exhibited selective cell
adhesion and proliferation of 3A6 cells (Human MSCs)
2
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than that of loss modulus (G'') indicating the gels are elastic
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DOI: 10.1039/D0TB01147A
rather than a viscous one (Fig. 1B and Fig. 1C). The elasticities of
the gels are varied depending on the pH of the hydrogel. At high
1
pH (10.2), a weak gel with G' of 6.75×10 Pa and as the pH of the
hydrogel decreased to 7.2, the storage modulus of the gel
3
increased to 1.25×10 Pa, however, further decrease in pH (4.7)
4
showed a dramatic increase of storage modulus to 7.08×10 Pa
was observed due to the formation of insoluble aggregates (See
Table. 1 for details).
Transmission electron microscope (TEM) was used to determine
the self-assembled nanostructures within the hydrogels at
various pH and concentrations. From the TEM images,
formation of thin nanofibers at 2.0 wt% under basic pH with
uniform diameter of 6±1 nm in width was observed. Once the
hydrogel‘s pH slightly decreased to 8.1 by adding dilute HCl, co-
existence of flat nanobelts and nanofibers were formed. At a pH
of 7.2 , a dense network of twisted nanobelts and fibers with
an average diameter of 52±4 nm and 5±1 nm in width was
observed and at pH below 3.7, the hydrogel was completely
disrupted and only clusters of thick and twisted nanobelts (Fig.
Fig. 1 A) Optical images of the self-supportive hydrogels (2.0 wt% ) with pH range from
2.0 to 3.7, and representative frequency sweep for the hydrogels with B) pH 7.2 to 4.7
and C) pH 10.2 to 8.1 respectively. G' (storage modulus) and G'' (loss modulus).
1
2
) was predominantly observed with slight increase in width
63±5 nm). The physical properties of the TPE-YY hydrogelator
and found that 0.75 wt% is the minimum gelation concentration at 2.0 wt% with varied pH were tabulated in Table 1. It is also
Fig. S1B). interesting to note that, the density of the nanobelts were
(
(
Since the TPE-YY material shown to be an effective hydrogelator decreased as we dilute the hydrogel sample to 500 µM (Fig. S3)
at broad range pH and concentrations. We further tested the with fibers as dominant. Thus, we conclude that as the
viscoelastic property of the hydrogels using oscillatory concentration increases, the formation of nanobelts exceeds at
rheometry, which is a fundamental requirement in tissue certain threshold which results in the formation of
38
regenerative applications. We performed frequency sweep supramolecular hydrogel. However, Ulijn and co-workers have
experiments of the hydrogels at 0.1 to 100 rad/sec with a strain detailed the effect of pH on the self-assembly mechanism of
a
of 0.8%. The storage modulus of the gels at 0.75 and 1.0 wt% aromatic dipeptide hydrogelator induced by pK shifts that
1
39
was found to be around 4.13×10 Pa which is not sufficient to undergo structural changes. Our perusal of literature on TPE
support the mass of the cell based on rheological experiments capped peptides demonstrate that this could be the first
(
37
Fig. S2). For any biological conditions the materials have to be findings to form hydrogel with broad pH range with tunable
designed to form hydrogel at physiological conditions and with nanostructures from nanofibers to twisted belts at varied pH.
a minimum storage modulus of G'>100 Pa.27,37 However, the We then investigated the self-assembly properties using various
gels formed at 2.0 wt% having storage modulus (G') greater trigger mechanisms such as heat-cool and by the addition of
Fig. 2 Representative negatively stained TEM micrographs of TPE-YY hydrogel at 2.0% w/v, at various pH ranging from pH= 12.0 to 3.7 units. pH=12.0-9.1 (entangled nanofibers),
pH= 8.1 (nanofibers and flat nanobelts), pH= 7.2 (twisted nanobelts and nanofibers), pH= 5.7-3.7 (thickened twisted nanobelts). scale bar: 50 nm
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twisted fibrils and nanotubes were observed.47 So we believe
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DOI: 10.1039/D0TB01147A
that in the case of TPE-YY, the self-assembly process might be
effected by the formation of NaCl, hence indicating the
significant role in promoting the nanofibers to twisted
nanobelts transitions, respectively. However, in the case of GdL
triggered hydrogel system, formation of NaCl is not impossible.
By the addition of excess amount of GdL resulted opaque
hydrogel and also gel syneresis was observed after 24 hour
ageing (Fig. S4). The rheological properties of the GdL triggered
hydrogel also suggests to be an elastic hydrogel Gʹ>Gʹʹ (Fig. S5C).
Further, in order to understand self-assembly kinetics in the
presence of GdL and acid-base trigger, we performed time
1
dependent rheology and time dependent H-NMR for the
Table. 1 Physical properties of TPE-YY at 2.0 w/v% with varied pH
pH
Appr.a
G, G
Pa)
ND
6.75×10 ,
.17×10
6.81×10 ,
.09×10
7.07×10 ,
.29×10
Nanostructures
diameter
[nm]
6±1
(
1
1
2.0
0.2
Sol
VG
Nanofibers
Nanofibers
1
5±2
1
1
1
9
.1
.2
TG
STG
OG
Nanofibers
5±1
1
1
1
8
Nanofibers and
Flat nanobelts
Nanofibers and
Twisted
7±2 and
50±9
5±1 and
52±4
1
1
3
Fig. 3 A) Negatively stained TEM image showing nanofibers with uniform diameter with
scale bar: 50 nm {Insets: optical image of the hydrogel formed at neutral pH} B) Time
dependent frequency sweep for the sample (2.0 w/v%) after adding GdL C) Time
7.2
1.25×10 ,
1.50×102
nanobelts
Only twisted
belts
3
1
5.8
4.7
OG
OG
3.13×10 ,
4
55±6
dependent H-NMR spectra for TPE-YY at 2.0 w/v% using GdL triggered self-assembly
2
.26×10
4
glucono-
has any influence on the self-assembly and hydrogelation
properties. Glucono- -lactone (GdL), a food sequestrant
-lactone (slow and mild pH change) to see whether it
7.08×10 ,
8.71×103
Twisted belts
58±11

*a) Sol: solution; TG: Transparent gel; VG: Viscous gel; STG: Semi-transparent gel;
OG: Opaque gel; TS: transparent solution; ND: Not detected
material, highly biocompatible and possess good solubility in
aqueous solution, undergoes slow hydrolysis to form gluconic-
40
acid by uniform reduction in pH. The amount of GdL addition hydrogels at 2.0 wt%. The time dependent rheological data
is one of the key requirement to achieve the desired pH.41,42 clearly shows that, G' is lower than G'' up to 5-10 minutes and
Surprisingly, the GdL addition resulted a very transparent after reaching to above 16 minutes G' is dominated over G'' (Fig.
1
hydrogel after 30 minutes under neutral pH (insets: Fig. 3A and 3B). Similarly, the time dependent H-NMR also complies with
Fig. S4). Briefly, to the 2.0 wt% solution of TPE-YY in 1M NaOH, slower rate of self-assembly as the proton peaks for TPE-YY
appropriate amount of GdL was added and left at room becomes broader due to the formation of self-assembled
temperature for 30 minutes to result a homogenous nanostructures after ~30 minutes (Fig. 3C). As the self-assemble
transparent hydrogel with pH 7.2. We then tested the process starts by slow hydrolysis of GdL making the solution
nanostructures associated with GdL method, interestingly, TEM neutralize with respect to time, the solution becomes viscous
images revealed a network of nanofibrous morphology (Fig. 3A and finally results a hydrogel which restrict the mobility of the
and Fig. S5). Therefore, different triggers resulted different molecules resulting in to broad signals. The proton signals
molecular packing for the formation of self-assembled related to gluconic acid did not broaden indicates that this small
nanostructures. We hypothesize that the self-assembly molecule was not involved in self-assembly mechanism with
41
1
mechanism with different triggers might be due to the peptide. Combining the time dependent rheological and H-
formation of sodium chloride (NaCl) in acid-base method. NMR study we anticipate that the gelation kinetics also may
Noticeably, there are well documented reports showing the play a role in determining the nanostructures. On the other
importance of various salts such as sodium chloride (NaCl), hand, the hydrogel formed via HCl addition gave broad signals
1
calcium chloride (CaCl
2
) and aluminium chloride (AlCl
3
) which in H-NMR within few seconds (typically < 10 seconds).
not only effects the self-assembly but also tune the morphology To gain a deeper insights in the molecular packing, we
4
3-46
1
1
of the peptide nanostructures.
For instance, Castelletto et performed one dimensional ( H-NMR) and two dimensional H-
1
1
al, studied the influence of NaCl concentrations on the self- H COSY and H-1H NOESY NMR techniques in DMSO-d
6
and
1
assembly of AAKLVFF and
concentrations of NaCl,

A
A


AKLVFF heptapeptides. At low DMSO-d
6
:Water mixtures. The H-NMR spectrum recorded in
solvent showed a doublet at 7.55 ppm, multiplets

AKLVFF showed highly twisted DMSO-d
6
fibrils while at higher salt concentrations co-existence of highly from 7.2 to 6.9 ppm, and a doublet at 6.6 ppm which
4
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of TPE-amide showed a significant shift from 7.55 ppm to 7.37
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DOI: 10.1039/D0TB01147A
ppm, notably, the remaining aromatic peaks showed a smaller
up-field shifts. Above this ratio, the compound is fully
1
aggregated and very broad H-NMR were obtained. Therefore,
we studied the molecular interaction using two dimensional
1
1
1
(
2D) NMR spectroscopic techniques such as H- H COSY and H-
1
H NOESY at 60% D
with pure DMSO-d
2
O:DMSO-d
6
solvent mixtures and compared
. In general, the H- H COSY used to see the
1
1
6
correlated protons which are in close proximity with each other.
Hence this technique helped us to identify and assign the
1
characteristic protons (See Fig. S6A and Fig. S6B). Similarly, H-
1
H NOESY spectrum is used to determine the intermolecular
interactions with in the molecule which are spatially close to
each other. A number of cross peak NOE correlations were
observed with in the assigned aromatic protons. As clearly
shown in Fig 4B, i-type protons correlated with j-type, l-type
correlated with k-type, h-type correlated with g-type and a-type
correlated with b-type protons illustrating the formation of
intermolecular aromatic interactions in Water:DMSO solvent
mixture. Whereas, these correlations were absent when
measured in simple DMSO-d (Fig. S6C). Thus 2D-NMR is an
6
important tool to identify the molecular interactions in the self-
assembled systems.
Circular dichroism (CD) and Congo red staining techniques were
employed for deeper understanding of molecular packing in self
assembled systems. From the Fig. 4C, the CD spectra at basic pH
(
12.0) is almost silent, upon aggregation (pH 10.0-7.0) by adding
dilute HCl, an intense CD signals were observed that
demonstating the aggregation induced circular dichroism
phenomena. Notably, at pH below 5.7, a significant reduction in
the CD signals was observed which can be ascribed to the
formation of insoluble aggregates. It should also be noted that,
there is a substantial change in the CD signals for nanofibers and
twisted nanobelts at pH 12.0 and pH 7.2, following a change in
molecular packing arrangement at varied pH conditions. Two
negative signals at 208 nm, 224 nm and a positive peak at 196
nm corresponding to the alpha-helical type of arrangement was
observed with the peptides in an aggregated state. In addition,
a clear negative Cotton effect with a negative part at λmax = 292
nm representing an ordered packing of π- conjugated
48,49
27
systems.
The Congo red staining for the hydrogel at 2.0
wt% at neutral pH showed a bright golden green birefringence
under polarized light microscope upon staining with Congo red
confirms the amyloid type of arrangements presents in the
molecular skeleton (insets: Fig. 4C). Fourier transform infrared
Fig. 4 A) Partial 1H-NMR recorded in D
using 300 MHz NMR instrument B) Partial H- H NOESY spectra with 60% D
using 500 MHz NMR instrument, C) CD spectra of 500 M at different pH D)
comparison of FTIR spectra in DMSO and in water at various pH
O: DMSO-d
solvent mixture from 10-60% D
2
6
2
O
1
1
2
O in DMSO-
(
FTIR) is a well known technique in determining the hydrogen
d
6
bonding interactions. The FTIR spectrum of the sample in DMSO
-1
-1
solution displayed a strong peaks at 1725 cm and 1645 cm
consistent with carbonyl streching for acid and amide peaks
corresponds to aryl rings of TPE and tyrosine groups, respectively (Fig. 4D, black line). At pH 12.0, the carbonyl
-
1
respectively. To examine the effect of aqueous solvent on self- streching band appeared at 1641 cm (Fig. 4D, green line), as
1
assembly, we measured the H-NMR spectra with different the pH lowered to 10.2, the molecules starts to aggregate and
-
ratios of D
2
O (10% to 60%) in DMSO-d
6
. As the concentration of then amide signals shifted towards lower frequency at 1633 cm
1
D
2
O increases for every 10%, the aromatic protons showed an
(Fig. 4D, blue line). At neutral pH, the corresponding signal for
up-field shift indicating strong hydrogen bonding interactions amide was broaden and shifted to much lower wavenumber to
-
1
and hydrophobic interactions between hydrogelators (Fig. 4A). 1610 cm (Fig. 4D, red line). Since FTIR is considered to be very
At 60% of D O in DMSO-d , the ortho positioned aromatic peak sensitive, these shift in peaks clearly indicates the extended
2
6
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-
5
the concentration of the solution is above 1×10 M there is a
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sudden rise in emission intensity due to the formation of
nanoaggregates. The plot of intensity versus concentration
reveals that cac as low as 10 µM (Fig. S9B). Since the TPE is well
known for its AIE characteristics, therefore we recorded the
water fraction in various ratios of DMSO-water solvent system
at 50 µM concentration. In pure DMSO, there is no emission was
seen when exited at λex =310 nm. The similar features were
continued with DMSO–water mixtures of water fractions (fw)
up to 80%. The emission intensity increases significantly when
the water fraction is above 80% and reaches the highest at 99%
water fraction with an emission maximum at λem = 455 nm (Fig.
5
C). The plot of relative emission intensity versus water was
shown to increase 60 folds at 99% water fraction, when
compared to pure DMSO (Fig. 5D).
To examine whether TPE-YY hydrogelator is a unique
biomaterial in regenerative medicine and tissue engineering
applications, we further studied cell adhesion and proliferation
of human mesenchymal stem cells (3A6) and mouse fibroblast
cells (L929). Therefore, we initially tested the in vitro
cytocompatibility of the peptide solutions using a colorimetric
assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT assay). The cells were cultured for 24 hrs and
Fig. 5 Fluorescence emission spectra for TPE-YY hydrogelator at A) different pH of the
solution with 500 µM concentrations B) Normalized PL spectra C) emission spectra in
DMSO/water mixtures (50 µM) {Insets: solid state fluorescence image} and D) The plot
of relative fluorescence intensity (I/I
0
) versus the water fraction {Insets: optical images
of the solutions with varied water content}
hydrogen bonding interactions between the hydrogelator then exposed with TPE-YY at the concentration ranges from 10-
1
5
molecules. From the powder X-ray diffraction (Fig. S7) 200 µM for 3 days, the experimental results revealed that cell
experiments the xerogels having a d-spacing of 4.26 Å at survival ratio of both the cell line was above 85 % at high
o
o
2
θ=20.5 and 3.24 Å at 2θ=27.4 which corresponds to concentration 200 µM even after the three-day culture (Fig. 6A
hydrophobic and hydrogen bonding interactions which is and Fig. 6B). In addition, we also evaluated cell viability assay
consistent with FTIR and NMR studies. No significant peaks with gel extraction medium (hydrogel leachable). TPE-YY
were observed from the lyophilized samples at pH 12.0. hydrogel leached with growth medium and then exposed to 3A6
Therefore, we deduce that the formation of self-assembled and L929 cells over the period of 3 days. Extraction medium
nanostructures of TPE-peptides are due to hydrophobic results displayed high cell viability that is above 95 % (Fig. 6C
interactions of TPE groups and also the extended hydrogen and Fig. 6D) suggesting that TPE-YY is nontoxic to both the cell
bonding interactions between peptides.
lines which can be considered as scaffolding material for tissue
We further investigated the photophysical properties at
variable pH and concentrations. The absorption spectra for TPE-
YY was found at λabs=310 nm (Fig. S8). To measure the
fluorescence
emission
at
varied
pH, we
used
photoluminescence spectroscopy (PL) and the stock solutions
were prepared at 5 mM concentration in 1M NaOH solution.
These stock solutions were then diluted to 500 µM with a series
of pH adjustments by adding dilute HCl from pH 12.0 to 3.0.
From Fig. 5A at basic pH (12.0), very low emission intensity was
observed when exited at λex=310 nm due to the free rotation of
phenyl rings of TPE, as the pH of the solution reduced to 7.2 the
RIR process becomes active and the emission intensity
dramatically increases and reaches maximum, further decrease
in pH 3.0 showed a steep drop in emission intensity due to
formation of precipitates (Fig 5A, insets: optical images of
solutions). In Fig. 5B the normalized PL at varied pH was plotted,
which confirms that with decrease in pH a slight red shift from
emission maxima λem= 455 nm to 465 nm was seen which
concludes that TPE-YY experiences more hydrophobic
-
interactions at acidic pH. We then quantitatively estimated the
critical aggregation concentration (cac) process by measuring
emission intensities at varied concentrations ranging from
Fig. 6 Cell viability of TPE-YY using MTT assay up to 72 hours at varying concentrations
of peptide solutions. A) 3A6 (hMSCs), B) L929(Mouse fibroblast) and 2.0 wt% Hydrogel
leachable C) 3A6 D) L929 cells. [*; P<0.05]
-9
-4
1
.0×10 M to 6.0×10 M in water (Fig. S9A). We noticed that if
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View Article Online
DOI: 10.1039/D0TB01147A
Fig. 7 Optical microscopic images for the cell adhesion experiments on gel coated surface after 24 hrs culture. left side images: Tissue culture plate; B, E, H, and K are the cellular
images of different cell lines seeded on TPE-YY coated hydrogel using acid-base method (B’, E’, H, and K’ are the representative magnified images); right side images of cells are
seeded on GdL triggered hydrogels. All hydrogels were prepared at physiological pH {Scale bar 100 µm}
engineering. Cyto toxicity of hydrogel leachable of TPE-YY (GdL) (Fig. 7B and 7E). Cells grown on TPE-YY hydrogel coated surface
were also tested with 3A6 and L9292 for 3 days, results suggest (nanofibers) showed poor cell adhesion and proliferation with
that these materials are non-toxic (Fig S10). Considering the both the cell line (Fig 7C and 7F) after 24hrs of culture. To
non-toxicity and high stability of TPE-YY hydrogel in culture further check the selectivity, we used two cancer cell lines (HeLa
medium, we further explored the proliferation and adhesion and MCF-7 cells) and grown on top of the hydrogels to study the
properties of 3A6 and L929 cells grown on TPE-YY hydrogel cell adhesion and growth behavior. The morphological
coated surfaces. The 3A6 cells were seeded at the density of appearance suggests that, cells were not adhered and thus
4
5
x10 cells on non-tissue culture plate pre-coated with TPE-YY remained spherical after 24 hrs of culture (Fig., 7H, 7K, 7I and
hydrogels (nanobelts and fibers) and the tissue culture plate 7L) in comparison with tissue culture plate (Fig 7G and 7J). The
o
(
TCP) used as control. The cells were then incubated at 37 C and zoomed-in images of the cells seeded on TPE-YY coated
5
2
% CO atmosphere for 24 hrs and 72 hrs. The morphology of substrates were represented in Fig. 7B’, 7E’, 7H’, and Fig. 7K’
adhered cells was then imaged with optical microscopy. 3A6- respectively. Further, it was suggested to measure the cell
cells grown on TPE-YY coated hydrogels substrate (nanobelts) circularity for quantitative analysis of the images to show the
displayed well adhesion with spindle shape morphology and differences in morphologies between samples of different cell
extended conformation with almost similar cell morphology of lines. Therefore, we measured cell circularity with ImageJ
those seeded on tissue culture plate (Fig. 7A and 7B) after 24 software. Cell circularity index showed that, 3A6 cells seeded on
hrs of culture. Optical microscopic images of 3A6 cells at 72 hrs TPE-YY hydrogel (acid-base method) coated surface displayed
showed that cells were proliferated well on the aforementioned less circularity which is similar to cells grown on TCP Fig. S12.
substrate (Fig S11B). Contrastingly, L929 cells grown on TPE-YY Whereas, L929, MCF-7 and HeLa cells grown on this hydrogel
hydrogel coated surface (nanobelts) does not adhered well and showed more circular, which also implies that 3A6 cells
the cell morphology was remained as spherical in shape after 24 selectively grown and spared on TPE-YY hydrogel. Thus, we
and 72 hrs culture (Fig. 7E and S11D) implying TPE-YY can be conclude from the cell adhesion and proliferation results of 3A6
considered as selective cell adhesive material for 3A6 over L929 demonstrate that TPE-YY hydrogel could act as promising
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The combined spectroscopic characterizations support the
View Article Online
DOI: 10.1039/D0TB01147A
influence of pH and gelation kinetics that play a crucial role in
self-assembly with tailored nanostructures. Furthermore, the
TPE-YY dipeptide hydrogel described here is potentially
biocompatible towards 3A6 and L929 cells for cell-imaging.
Interestingly, the nanobelts of TPE-YY displayed selective cell
adhesion response for 3A6 cell in comparison with L929 and the
cancer cells (HeLa, MCF-7), while the nanofibers showed poor
cell responses representing nanobelts as an excellent scaffold
for stem cell-based therapies. Therefore, the current work may
provide new insights for the design of chemical structure, pH
and morphology relationships to develop future bio-materials
in tissue engineering applications.
Fig. 8 Confocal fluorescent microscopic images of 3A6 and L929 cells at 50 µM
concentrations for 90 minutes of incubation time. Above: 3A6 and Below: L929 cells,
respectively. {Scale bar: 20 µm}
Experimental
Reagents and Materials
Diphenylmethane and 4-bromobenzophenone was purchased
from Alfa-aesar, n-BuLi (2.5M in Hexane) was purchased from
Rookwood, HBTU from Matrix scientific, Diisopropylethylamine,
biomaterial for stem cell based therapies. In contrast, when the
3
A6 cells seeded on the nanofibers resulted from the GdL
hydrogelation method showed lower cell density and slower
rates of proliferations when compared with nanobelts (Fig. 7C,
piperidine and Trifluoroacetic acid from Alfa-aesar. Fmoc-
L-Tyr-
7
F, 7I, and 7L), indicating that nanobelts promote the extensive
(
OtBu)-OH from Carbosynth limited (UK), Anhydrous grade
cell adhesion and proliferation of 3A6 cells on hydrogel.
Previously it was demonstrated that the nanostructures with
the diameter ~ 50 nm will enhance cell adhesion response.50-52
The nanostructure morphology and the hydrogel was not
disrupted even after the addition of culture medium (DMEM)
on top of the hydrogel (for TEM and optical images see Fig. S13
and S14). Based on the above experiments our designed
hydrogels are highly viable and selective towards 3A6 cells.
Since TPE is an archetypical AIE material, it was suggested to
perform in vitro bio-imaging. In vitro cellular imaging of TPE-YY
was studied using 3A6 and L929 for 90 minutes of incubation
time. The cells were incubated with the lower concentrations of
solvents were purchased from Sigma Aldrich and used as
received.
General procedure for synthesis of compound TPE-YY:
The peptide derivative TPE-YY were synthesized by a standard
solid phase peptide synthesis (SPPS) method using 2-chlorotrityl
chloride resin as a solid phase substrate. Briefly, the resin (1.2
g, 2.0 mmol) was suspended in anhydrous dichloromethane and
allowed to swell the resin for 30 minutes under stirring in
nitrogen atmosphere. Then the Fmoc-Tyr-(Boc)-OH (3.0 mmol)
and N,N-diisopropylethylamine (5.0 mmol) were dissolved in an
appropriate amount of anhydrous dimethylformamide and was
added to resin. It was reacted for 60-90 minutes to allow the
amino acid to be attached to the resin. The Fmoc protecting
group was removed by reaction with a solution of 20%
piperidine in dimethylformamide for 30 minutes and repeated
twice (2 minutes each time). Then, the second batch of Fmoc-
Tyr-(Boc)-OH (3.5 mmol) was coupled to resin bound amine
o
TPE-YY i.e., at 50 µM for 90 minutes at 37 C in the culture
medium and then fixed with para formaldehyde, the cellular
images were taken with confocal microscopy with a 405 nm
laser excitation and the fluorescent signals were collected and
imaged. The confocal fluorescent microscopy images show that
the TPE-YY was accumulated in cytoplasm of both the cell lines
with homogeneous distribution excluding nuclei were seen with
bright blue fluorescence (Fig. 8). The low cellular toxicity with
bio-imaging results manifest that TPE-YY behaves as a good
fluorescent bio probe in cellular imaging applications.
using
coupling
agent
benzotriazole-N,N,N',N'-
tetramethyluronium hexafluorophosphate, HBTU (5.0 mmol)
and N, N-diisopropylethylamine (5.0 mmol) in an appropriate
amount of anhydrous dimethylformamide, and allowed to react
for 60-90 minutes. Then again Fmoc group was deprotected by
reacting with
a
solution of 20% piperidine in
dimethylformamide for 30 minutes, and washings was repeated
twice (2 minutes each time). Lastly, N-terminal capping agent,
tetraphenylethylene acid TPE-Acid (4.0 mmol), HBTU (3.0
Conclusions
In conclusion, our demonstration on tetraphenylethylene mmol) and N,N-diisopropylethylamine (7.5 mmol) was
based dipeptide (TPE-YY) as a unique short peptide fluorescent dissolved in an appropriate amount of anhydrous
hydrogelator exhibited tunable self-assembly into various dimethylformamide, and was added to resin bound amine. This
nanostructures and thus transformed to supramolecular was allowed to react for overnight under nitrogen condition.
hydrogel with broad range of pH (3.7 to 10.2). From the The reaction was washed with DMF, DCM, MeOH, and Hexane
understandings of our previous study, replacement of aliphatic three times with each solvent. Finally, the deprotection of resin
amino acids with two tyrosine residues provided extended from the peptide was carried with 30% trifluoroacetic acid (TFA)
hydrogen bonding and hydrophobic interactions that drive the in dichloromethane for 3 hrs. The TFA solution was air dried and
self-assembled nanostructures into supramolecular hydrogel. precipitated by the addition of cold diethylether, filtered and
8
| J. Name., 2012, 00, 1-3
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Page 9 of 12
Journal of Materials Chemistry B
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Journal Name
ARTICLE
washed with diethylether and dried under vacuum to afford the Sweep mode is “log” and the experimental temperature
View Article Online
1
o
DOI: 10.1039/D0TB01147A
desired TPE-YY (550 mg) as off-white solid. H-NMR (300 MHz, maintained at 25 C.
DMSO-d , δ): 2.71-2.98 (m, 4H), 4.30-4.35 (m, 1H), 4.52-4.65 (m Congo Red Staining:
1H), 6.62 (d, J =8.4 Hz, 4H ), 6.90-7.01 (m, 10H), 7.02-7.19 (m,
1H), 7.58 (d, J =8.4 Hz, 2H), 8.18 (d, J =8.1 Hz, 1H, NH), 8.42 (d,
, δ): 36.0, 36.1,
3.9, 55.1, 114.9, 115.1, 126.7, 126.8, 126.9, 127.1, 127.2,
6
,
1
To a freshly prepared saturated sodium chloride ethanol
solution (80%) was added Congo red stain and the resulting
solution was vortexed and filtered. Congo red solution was
pipetted onto a glass microscope slide, and then the 2.0 wt%
hydrogel was placed underneath the surface of the Congo red
solution and stained for approximately 1-2 min. After the excess
Congo red solution was removed by blotting, images of the
sample placed between crossed polarizers were obtained with
ZEISS microscope.
J =8.4 Hz, 1H, NH); 13C-NMR (75 MHz, DMSO-d
6
5
1
1
1
27.4, 127.9, 128.0, 128.1, 128.5, 129.1, 130.2, 130.6, 131.9,
39.9, 141.5, 142.9, 142.93, 143.0, 146.3, 146.4, 155.74, 156.0,
65.94, 171.7, 172.9. High resolution mass HRMS: calcd.
m/z:702.2730, obsvd: 701.2735 [M-H].
Preparation of Hydrogel:
Cell viability (Gel extraction medium):
The gelation experiment was performed by carefully weighing
The TPE-YY hydrogels were prepared under sterile conditions in
a 15mL sterile tube and pH was then adjusted to 7.4. TPE-YY
hydrogel was then leached by Dulbecco’s modified Eagle’s
medium (DMEM) low glucose (HyClone, Cat # SH30021.02)
containing 10% fetal bovine serum (HyClone, Cat #SH30071)
supplemented with Penicillin/Streptomycin 100 units/mL
4
.0 mg of hydrogelator TPE-YY in a 2 mL vial (diameter 10 mm)
with a screw cap and the addition of 0.20 mL of the solvent and
adjusting pH with 1 M HCl solution to form gel. For GdL method,
4
.0 mg of TPE-YY was dissolved using 1 M NaOH in 0.20 mL, the
appropriate amount of GdL was added and stirred gently using
spatula and left at room temperature until become a gel.
Gelation is considered to have occurred when the material is
obtained does not show gravitational flow (confirmed by
inverted test tube method for 5 min).
(
(
HyClone Cat #SV30010) for human mesenchymal stem cell line
3A6 cells) was placed on top of hydrogel and incubated for 3
days under a humidified atmosphere of 5% CO
mouse fibroblast L929 cells, Eagle's Minimum Essential Medium
EMEM, ATCC, Cat # 30-2003) containing 10% Horse serum
2
at 37°C. For
Instruments Used:
(
1H-NMR and 13C-NMR spectra were characterized using a 300 supplemented with Penicillin/Streptomycin 100 units/mL
MHz Bruker instrument with deuterated dimethyl sulfoxide (HyClone Cat #SV30010) was placed on top of hydrogel and
1
1
1
1
(
6 2
DMSO-d ) at room temperature. H- H NOESY and H- H COSY incubated for 3 days under a humidified atmosphere of 5% CO
were recorded in 500 MHz variant instruments. Mass spectra at 37°C. The 3A6 and L929 cells were seeded on to a 24 well
4
were recorded using a Q-TOF Micro MS spectrometer. FTIR tissue culture plate at a density of 2×10 cells in each well and
spectra were measured using a Perkin Elmer spectrum 100 then incubated at 37
2
℃ under 5% CO atmosphere for 24 hrs.
-
series spectrometer and were collected at a resolution of 4 cm After 24 hrs of incubation, the medium was removed, and cells
1
using a detector by averaging scans (20 scans). UV-Vis were then incubated with gel extraction of TPE-YY for 24 hrs, 48
absorption and circular dichroic spectra (CD) were measured hrs, and 72 hrs. The medium was replaced at each time point
with a spectropolarimeter (JASCO J-815, cell length 1 mm, 25 °C) with fresh medium containing MTT reagent 0.5 mg/mL final
from 190 to 450 nm and the concentration of peptides for UV concentration and incubated for another 4 hrs, then MTT
and CD measured from 250m to 5000m. Fluorescence reagent was removed and DMSO was added to dissolve the
emission spectra were obtained using a Hitachi F-4500 formazan crystals, the 100 µL of each 24 well plate was
fluorescence spectrometer at an excitation wavelength of transferred to 96-well plate and the optical density of resulting

=310 nm with the at different concentrations and pH.
solution was measured at 595 nm using TECAN Infinite F50
microplate reader. Cells without the treatment of gel extraction
were used as the reference.
Transmission Electron Microscopy (TEM):
The images were obtained using a Hitachi HT7700 transmission
electron microscope at an accelerating voltage of 100 kV.
Cell viability (peptide solutions from 10-200µM):
Hydrogels were applied directly onto 200 mesh carbon-coated Cell viability assay was performed using a colorimetric assay
copper grids. An excess amount of the hydrogel was carefully (MTT). The 3A6 and L929 cells were seeded at density 2×104
removed by capillary action (filter paper), and the grids were cells per well in a 24-well tissue culture plate and cells were then
then immediately stained with uranyl acetate for 30 seconds. incubated for 24 hrs at 37
2
℃ under 5% CO atmosphere. After
Excess stain was removed by capillary action, and the grids were 24 hrs incubation, the medium was replaced with freshly
allowed to air dry for 24 hrs.
Rheological tests:
prepared medium containing samples at different
concentrations (10 to 200 µM) for the period of 24, 48 and 72
hrs. The medium was then replaced at each time point with
fresh medium containing MTT reagent 0.5 mg/mL final
concentration and incubated for additional 4 hrs, then MTT
reagent was removed and DMSO was added to dissolve the
formazan crystals, the 100 µL of each 24 well plate was
transferred to 96-well plate and the optical density of resulting
Rheological tests were conducted on TA instrument. 25 mm
parallel plate was used during the experiment. 200 µL of
hydrogel sample was placed on the parallel plate. Angular
frequency sweep test: Test range (0.1 to 100 rads frequency,
strain = 0.8 %), 13 points per decade. A solvent trap was used to
keep the sample hydrated and the chamber filled with water.
-1
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8
9
V. Kayser, D. A. Turton, A. Aggeli, A. Beevers, G.VDiew. RAretiicdle, OGn.linSe.
solution was measured at 595 nm using TECAN Infinite F50
microplate reader. Cells without the treatment of gel extraction
Beddard, J. Am. Chem. Soc. 2004, 126, 336.
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G. B. Fields, Curr Protoc. Protein. Sci, 2002, Chapter 18, Unit-
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.
1
8.1.
1
1
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3
3
1
A6 or L929 cells were seeded in a 35-mm petri dish at density
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0% (v/v) FBS/HS and penicillin/streptomycin (100 μg/mL)
1
3
respectively at 37 °C in a 5% CO2 atmosphere after 12 hrs
incubation. The culture medium was then removed and cells
were exposed with TPE-YY at 50 μM concentration 90 mints,
harvested cells were then washed with 0.1 mM phosphate-
buffered saline (PBS) and then cells were fixed with 4 %
paraformaldehyde, post fixation cells were washed with PBS
and then sample were examined under a Multiphoton Confocal
Microscope System (TCS-SP5-X AOBS , Lecia , Germany) 63 X oil-
1
0467.
1
4
K. McAulay, B. Dietrich, H. Su, M. T. Scott, S. Rogers, Y. K. Al-
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1
8
1
2
2
2
9
0
1
2
Cell adhesion:
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3
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o
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