Biocompatible small peptide super-hydrogelators bearing carbazole functionalities

Article abstract of DOI:10.1039/c5tb00067j

For the first time we have introduced carbazole capping groups onto two short peptide sequences, namely a diphenylalanine dipeptide and a glycine-diphenylalanine tripeptide, giving compounds 1 and 2, respectively. Both molecules form hydrogels at low concentrations, as low as 0.03% (w/v) for 1 - well within the range of supergelators. Both gelators are composed of small ca. 2 nm thick molecular fibres, as elucidated by AFM and are non-cytotoxic towards HeLa cells at concentrations which are at or above their minimum gelation concentration.

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Biocompatible small peptide super-
hydrogelators bearing carbazole functionalities
Cite this: DOI: 10.1039/x0xx00000x
Adam D. Martin,*a Andrew B. Robinson,a Pall Thordarson*a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
For the first time we have introduced carbazole capping molecular fibres approximately 2 nm thick and while it has often been
groups onto two short peptide sequences, namely suggested that it would be useful for medical applications, it does
diphenylalanine dipeptide and
glycine-diphenylalanine appear to show some cytotoxicity in vitro at elevated concentrations.18
a
a
tripeptide, giving compounds 1 and 2, respectively. Both Typically, diphenylalanine-based gelators require low pH in order to
molecules form hydrogels at low concentrations, as low as form a hydrogel, however the addition of a less hydrophobic glycine
0.03% (w/v) for 1 – well within the range of supergelators. residue has been shown to raise the pH at which gelation occurs,19
Both gelators are composed of small c.a. 2 nm thick rendering the hydrogel more biocompatible. This has been observed
molecular fibres, as elucidated by AFM and are non-cytotoxic for multiple capping groups.20
towards HeLa cells at concentrations which are at or above
their minimum gelation concentration.
The selection of aromatic capping group also plays a significant
role in the properties exhibited by the resultant hydrogel. For short
peptides, an aromatic capping group is often required to drive
gelation due to hydrophobic and π-stacking interactions. The most
common capping group is Fmoc, however other capping groups are
based upon different aromatic molecules such as anthracene,21
naphthalene,22 perylene23 and pyrene.24 These capping groups all
provide the hydrophobic interactions necessary, and in the case of the
last two, impart fluorescence upon the resultant gels. Heterocycles
have also been employed as effective capping groups, with examples
such as phenothiazine25 and spiropyran reported.26 Recently we
synthesised an indole-capped dipeptide that displays extremely high
values for its storage modulus, we theorised due to the hydrogen
bonding potential of the indole moiety.27
Introduction
Biomimetic materials have recently attracted large amounts of
interest due to their potential applications in the fields of drug
delivery,1,2 as cell scaffolds3 and for tissue engineering.4,5 Hydrogels
are an important class of biomimetic materials, as they are able to act
as mimics for the extracellular matrix, which promotes cell adhesion,
differentiation, growth and proliferation.6-8 Unlike polymer hydrogels,
which are typically formed through the incorporation of water-
soluble groups on a synthetic polymer backbone,9,10 supramolecular
hydrogels offer an advantage in that they are reversible in nature,
owing to the non-covalent interactions that comprise the gel network.
Peptide hydrogels are an important subset of supramolecular
hydrogels which are promising materials for applications within the
human body due to them being composed of naturally occurring
amino acids.11-13
Carbazole can be thought of as an analogue of the fluorene
moiety that is present in the Fmoc capping group so widely used in
peptide synthesis. Fluorene-capped dipeptides not containing the
carbamate linkage (whereby peptides are connected via an amide
linkage instead) have been synthesised and tend to be slightly less
robust compared to their Fmoc analogues.28,29 Herein we report the
first examples of carbazole capped peptides, which were achieved
The variety of peptides that can be used for biological applications
is almost limitless, however short peptides are attractive targets due
to their ease of synthesis, the scalable nature of this synthesis and
gelation ability. Numerous 9-fluorenylmethyloxycarbonyl (Fmoc)-
capped dipeptides have been reported in the literature,14-16 however
perhaps the most well-known example is Fmoc-diphenylalanine
(Fmoc-Phe-Phe).17 This peptide has been shown to self-assemble into
through attachment of 9H-carbzole acetic acid to diphenylalanine (-
Phe-Phe, Fig. 1a) and glycine-diphenylalanine (-Gly-Phe-Phe, Fig. 1b)
amino acids.
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Experimental
from 300 – 350 nm that are opposite to each other but similar effects
have been observed for peptides containing Gly-Phe-Phe.32 ATR-IR
9H-carbazole acetic acid was synthesised according to a modified
spectra of hydrogels of
1
and
2
are almost identical, with only minor
and
literature procedure.30 –Phe-Phe and –Gly-Phe-Phe amino acid
residues were synthesised using standard solid phase peptide
synthesis techniques (see ESI for synthesis and characterisation data
changes in the fine detail of some peaks (Fig. 1d). Hydrogels of
1
2
both contain peaks in the amide I (1600 – 1700 cm-1) and amide II (1510
– 1580 cm-1) regions, with the peak position in the amide II region
confirming the β-sheet secondary structure obtained from CD
measurements.
for capping group and capped peptides), with
1 and 2 being obtained
in 48% and 49% yield, respectively. Both compounds were then
purified using semi-preparative high performance liquid
chromatography before further characterisation.
Results and Discussion
The gelation properties of both
purification, initially using the vial inversion method.
hydrogels via a pH switch mechanism only, whereas is able to form
1
and
2
were investigated after
1
forms
2
hydrogels either through pH switching, temperature switching in a 1
mM PBS buffer (pH 7.2) solution or through solvent switching,
whereby an alkaline solution of
2 is diluted using DMEM, instantly
forming a self-supporting hydrogel. It should be noted that for pH and
solvent switching, sonication is used to initiallydisperse the gelator,
forming a homogenous sol in all cases, before changing the solvent
environment to result in gelation.
low concentration of 0.03% (w/v), making this compound
supergelator and close to the lowest reported minimum gel
concentration for peptide hydrogel. In comparison to the
minimum gel concentration for is 0.1% (w/v), which is low, and
indicates that both and have the ability to form ordered
nanofibrous networks at extremely low concentrations. Given that the
Fmoc analogues of and do not form hydrogels at such low
concentrations (in fact, no gelation was reported for the Fmoc
analogue of
using an enzymatic cleavage mechanism),31 it can be
1 forms hydrogels at the remarkably
a
a
1,
2
1
2
Fig. 2 AFM at different scan sizes, clearly showing fibre morphology of
and (c, d). Both hydrogel samples were prepared at a concentration of 0.1%
(w/v) and imaged under ambient conditions.
1 (a, b)
2
1
2
2
In addition to investigating the secondary structure of hydrogels
and , the morphology of the gel network formed by and was
also investigated. It was found that in both cases, small, molecular
nanofibres comprise the gel network. For , an average fibre size of 1.7
inferred that these values observed are due to the selection of the
carbazole capping group.
of
1
2
1
2
1
± 0.3 nm was measured, which corresponds closely to the measured
individual fibre size for Fmoc-Phe-Phe of approximately 2 nm. This
implies that gels of
1 are composed of molecular fibres, much like the
analogous Fmoc--Phe-Phe. For hydrogels of 2, incorporation of a
glycine residue serves to increase the fibre diameter to 2.2 ± 0.4 nm.
This is smaller than the 20 nm fibre size reported for the gelator
napthalanine-Gly--Phe-Phe,20 and smaller than the
5 nm fibres
measured in the Fmoc analogue of 2 (Fig. S16). This small fibre size
persists even at higher concentrations and using different deposition
methods (Fig. S6 and S12), indicating that the carbazole capping
group has a propensity for forming small, molecular fibres.
Fig. 1 Structure of carbazole capped peptides
hydrogels of and (c) and amide I and II region of the ATR-IR spectrum for 1%
(w/v) hydrogels of and (d)
1 (a) and 2 (b), CD spectra of
Despite the similarities of
1 and 2 in terms of fibre size, their
1
2
morphology is actually very different. It can be seen for hydrogels of
1, the nanofibres observed are quite disjointed and not continuous
(Fig. 2a). In this way, the system is more like a highly branched
network of nano-fibrils. These fibrils do not possess a uniform length,
and can be seen to intertwine and intersect with each other (Fig. 2b).
1
2.
The secondary structure of
1 and 2 was then probed using circular
dichroism, with both hydrogels displaying a β-sheet structure (Fig. 1c).
Due to difficulties in quantifying gel dilution, ellipticities per residue
cannot be compared, however, the position of the minima in the CD
For hydrogels of
2 however, this highly branched system of fibrils is
replaced by fibres which are largely continuous, and although some
fibre ends can be observed, the majority of the fibres imaged have
lengths in the order of micrometers, Fig. 2c. Less branching is
observed in this system; however fibres which intersect with each
spectrum are found at 221 and 216 nm for
1 and 2, respectively,
typical for hydrogels bearing the diphenylalanine motif. It is
interesting to note that the hydrogels display features in the spectrum
2 | J. Name., 2012, 00, 1-3
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other are still present. Perhaps the most striking difference though is hydrogels prevented imaging at higher concentrations of gelator. It is
the handedness that is clearly visible for these fibres. The vast also evident from Fig. 4 that cell viabilities are very similar for both
majority of the fibres that can be seen in Fig. 2d are left handed, and , meaning that it is most likely the selection of capping group,
1
2
without any evidence for right handed fibres being present. The fact not the alteration of an amino acid residue that is responsible for any
that this change in morphology is brought about by the addition of an cytotoxic effects observed. Based upon the data in Figure 4, we can
achiral glycine residue in
2
is rather unexpected.
estimate IC50 values for 1 and 2 of 0.18 and 0.10% (w/v) respectively. In
both cases, this value is at or above than the minimum gel
concentration for these peptides. This makes both peptide gelators
good candidates for further study employing different cell lines and as
potential extracellular matrix (ECM) mimics or drug delivery vectors.
Fig. 3 Rheology of 1% (w/v) hydrogels of 1 (a) and 2 (b) performed at 25 °C. Error
bars indicate standard deviation from repeat experiments (n = 3).
Rheology studies were then undertaken to investigate whether
the difference in fibre morphology at the nanoscale resulted in a
difference the macroscopic rheological properties for hydrogels of
and . As can be seen from Fig. 3, this difference in nanoscale
structure results in large difference in gel strength, which is
1
2
a
commonly approximated using the values obtained for the storage
modulus G’. At 1% (w/v) and both gels exhibit linear behaviour over
Fig. 4 Cytotoxicity studies of
time period of 24 h.
1 and 2, performed on HeLa cells with n = 3, over a
the frequencies measured. Hydrogels of
1
display a storage modulus
. This represents
of 0.5 – 0.7 kPa, compared to 3 kPa for hydrogels of
2
Conclusions
a greater than 4-fold increase in gel strength due to the incorporation
of a glycine residue into the peptide structure. Rheology was also
performed at the minimum gel concentrations of both hydrogels (Fig.
In conclusion, we have synthesised two novel peptides bearing for the
first time a heterocycliccarbazole capping group. These short peptides
S5 and S11). In this case, hydrogels of
Pa, compared to 1 kPa for hydrogels to
low concentrations, hydrogels of are stiffer than those of
weak nature of hydrogels of is apparent from viewing the nanoscale
1
have a storage modulus of 150
. This confirms that even at
. The
differ by
a single amino acid residue, and form self-supporting
2
hydrogels at very low concentrations, and in the case of
1
,
2
1
concentrations which make it a supergelator. Both peptides exhibit a
β-sheet secondary structure and form nanofibres that are
1
structure – the discontinuous nature of the fibres, resulting in fibril
formation coupled with the highly branched nature of these fibrils.
These discontinuous fibrils mean that the persistence length
(essentially the length of aligned segments within a fibre) for these
hydrogels is low, and the storage modulus G’ has been shown to be
dependent on the square of persistence length.33 This helps explain
approximately 2 nm in diameter. Fibres of
and have a high proportion of junctions, resulting in a soft gel, which
has been confirmed by rheology. In contrast, displays longer, left-
1 are more discontinuous
2
handed nanofibres. Cytotoxicity studies on HeLa cells confirm that the
gels are well tolerated up to 0.1% (w/v), which for both peptides is at
or above their minimum gel concentration –in the case of
order of magnitude above its minimum gel concentration.
1, almost an
why the storage modulus values for hydrogels of
2 are so much
higher, because even though the fibre size is still small, the
persistence length of these fibres is much larger than for hydrogels of
Acknowledgements
1
.
We would like to thank the Mark Wainwright Analytical Centre
(UNSW) for access to instruments. We acknowledge the Australian
Research Council for Discovery Project Grant (DP130101512) an ARC
Centre of Excellence Grant (CE140100036) and a Future Fellowship to
PT (FT120100101) and support from the Commonwealth Science and
Industrial Research Organisation (CSIRO) to PT and ABR.
The size of the fibres observed for hydrogels of
1 and 2, coupled
with their relatively low values for G’ makes these materials excellent
candidates for extracellular matrix mimics. To this end, preliminary
cytotoxicity studies were undertaken using a HeLa cell line. It can be
seen that both 1 and 2 are well tolerated up to a concentration of 0.1%
(w/v). It should be noted that for both gelators, this is at or above their
minimum gel concentration. Cytotoxicity results were similar over 24
and 48 hours (Fig. S15), suggesting that the cell death observed may
be due to initial contact cytotoxicity, and is not a sustained process.
Notes and references
aSchool of Chemistry, The Australian Centre for Nanomedicine and the
ARC Centre of Excellence for Convergent Bio-Nano Science and
Technology, The University of New South Wales, Sydney, 2052, NSW,
Australia.
Images of cells seeded on top of hydrogels of
1 and 2 show some cell
spreading and proliferation (Figure S14), however the opacity of the
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DOI: 10.1039/C5TB00067J
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Two novel short peptides bearing a novel carbazole capping group form gels at concentrations as low
as 0.03% w/v and are biocompatible at or above their minimum gel concentrations.