Peptide-based hydrogen sulphide-releasing gels

Article abstract of DOI:10.1039/c5cc04883d

An aromatic peptide amphiphile was designed for delivery of the signaling gas H₂S. The peptide self-assembled in water into nanofibers that gelled upon charge screening. The non-toxic gel slowly released H₂S over 15 hours, and the presence of H₂S in endothelial cells was verified using a fluorescent H₂S probe.

Full text of DOI:10.1039/c5cc04883d

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Peptide-based hydrogen sulphide-releasing gels†
Jennifer M. Carter, Yun Qian, Jeffrey C. Foster and John B. Matson*
Cite this:DOI: 10.1039/c5cc04883d
Received 15th June 2015,
Accepted 11th July 2015
DOI: 10.1039/c5cc04883d
www.rsc.org/chemcomm
An aromatic peptide amphiphile was designed for delivery of the In contrast, few H2S-releasing materials have been prepared for
signaling gas H2S. The peptide self-assembled in water into nano- potential use in therapy: two polymeric H2S donors5 and one
fibers that gelled upon charge screening. The non-toxic gel slowly H2S-releasing metal–organic framework.9 While polymers and
released H2S over 15 hours, and the presence of H2S in endothelial other nanoparticle drug delivery systems may be useful for
cells was verified using a fluorescent H2S probe.
targeting specific tissues through systemic administration,
H2S-releasing gels that can be localized at a site of interest are
Despite its reputation as a foul-smelling, toxic pollutant, hydrogen better suited as injectable biomaterials capable of filling a cavity
sulphide (H2S) is a vital biological signaling gas (gasotransmitter) or encapsulating cells while delivering precise doses of H2S to an
produced endogenously by various enzymes.1 Within the past few area of interest.
years, efforts devoted to studying the biological roles of H2S, the
We recently reported on a family of small molecule H2S
enzymes that make it, and its therapeutic potential have expanded donors based on the S-aroylthiooxime (SATO) functional group,
rapidly,2 along with related efforts to improve its detection in vitro which releases H2S in response to thiol functionality.4e SATOs
and in vivo.3 While many biological studies on H2S are conducted are prepared by reaction of an S-aroyl thiohydroxylamine
using sulphide salts (NaHS and Na2S), injections of aqueous (Ar–C(O)S–NH2) (SATHA) with an aldehyde or ketone. H2S release
sulphide solutions lead to a rapid surge of H2S in the bloodstream is triggered by thiols such as cysteine, and release rates can be
followed by a rapid decline. To combat this uncontrolled delivery tuned by changing substituents on the SATHA ring, with half-lives
and more closely mimic the slow and sustained release of of release in the range of 8–80 minutes.
endogenous H2S, our group and others have reported on small
In this report, we describe the preparation of a self-
molecules that release H2S with controllable kinetics.4 However, assembling peptide designed to form an H2S-releasing gel.
despite this progress on small molecule H2S donors, very few Self-assembling peptide-based materials include a wide range
efforts have been made in the area of H2S-releasing materials.5
of peptide motifs that form one-dimensional nanostructures in
In addition to H2S, nitric oxide (NO) and carbon monoxide aqueous solution.10 Gel formation in the range of 0.5–2 wt%
(CO) are also classified as gasotransmitters. The study of NO peptide can often be triggered by charge screening through physical
biology has benefitted greatly from NO-releasing materials, entanglement of the extended nanostructures by pH changes or
including polymers, gels, microparticles, and others.6 The addition of salt. No chemical crosslinking is required, allowing for
localized release provided by these materials allows for site- in situ gelation of the materials and shear-thinning behaviour. These
specific delivery of the gas, which minimizes the required qualities, along with their inherent biodegradability and minimal
dosage and off-target effects that can result from systemic NO immunogenicity in most cases, make peptide-based gelators an
administration. In fact, NO-releasing materials have been studied attractive alternative to traditional polymeric hydrogels that require
widely in preclinical animal models for various applications.7 chemical crosslinking.
Additionally, CO releasing materials have also been reported,
One class of self-assembling peptides is the aromatic peptide
although they have not yet reached the stage of animal models.8 amphiphiles.11 Comprised of short oligopeptides with terminal
aromatic components, these small molecule gelators can be
Department of Chemistry and Macromolecules and Interfaces Institute, Virginia
designed to self-assemble in water into extended cylindrical
micelles or nanofibers.12 The aromatic groups in aromatic peptide
Tech, Blacksburg, VA, 24061, USA. E-mail: jbmatson@vt.edu
† Electronic supplementary information (ESI) available: UV-vis and fluorescence
amphiphiles have traditionally served a structural role—none
spectroscopy data, CMC plots, additional TEM, rheology, and H2S-release data,
have included reactive groups designed to release a bioactive
small molecule such as H2S.
experimental procedures, and characterization data for all new materials. See
DOI: 10.1039/c5cc04883d
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Fig. 1 (A) Conventional TEM image of peptide 3 cast from water (stained
with 2% uranyl acetate). (B) Frequency sweep rheology of peptide 3 at 1 wt%
in water gelled with CaCl2. Inset shows photograph of self-supporting gel.
Scheme 1 Synthesis of SATO-containing aromatic peptide amphiphile 3
from SATHA 1 and aldehyde-containing peptide 2.
solution of peptide 3 in water led to instantaneous gelation
Our design (Scheme 1) takes advantage of the inherent hydro- (Fig. 1B inset). As expected based on the TEM observations,
phobicity of the SATO moiety to serve as both a structural compo- addition of CaCl2 to a solution of peptide 2 did not result in
nent to drive self-assembly as well as a reactive functional group to gelation. The viscoelastic properties of the self-assembled peptide
release H2S. We envisioned that a SATO-functionalized aromatic hydrogel prepared from a 1 wt% aqueous solution of peptide 3
peptide amphiphile could be designed to form 1-dimensional were characterized by rheology (Fig. 1B). These experiments
nanostructures capable of gelation. Additionally, we anticipated that revealed that the gel was robust, with a storage modulus of
the SATO functional groups would be buried in the hydrophobic 320 Pa. Both the storage and loss moduli for peptide 3 remained
core of the nanostructures, thereby limiting their access to the steady independent of frequency up to 50 Hz.
cysteine trigger required for decomposition and potentially
extending their H2S-releasing capacity over hours to days.
To evaluate the self-assembled structure of the nanofibers,
we first measured the critical micelle concentrations (CMCs) of
The peptide design we envisioned included a hydrophobic peptides 2 and 3 using the Nile red assay. Both peptides had
SATO group attached to a short peptide that facilitated assembly CMC values of approximately 1 mg mLÀ1 (Fig. S6, ESI†). Next, we
into 1-dimensional nanostructures. In addition to aromatic stacking characterized peptide 3 by circular dichroism (CD), IR, UV-Vis,
interactions in the nanofiber core, many aromatic peptide amphi- and fluorescence spectroscopies. For all four characterization
philes also rely on b-sheet-type hydrogen bonding along the long axis techniques, experiments were performed at a concentration
of the nanostructure to stabilize the cylindrical assembly.13 Toward below the CMC (0.25 mg mLÀ1), above the CMC (10 mg mLÀ1),
this end, the peptide IAVE3 was synthesized under standard solid and on the gel (10 mg mLÀ1 gelled with CaCl2).
phase conditions using Fmoc chemistry. The IAV region was
Self-assembling peptides often have strong intermolecular
designed based on the propensity of certain residues to self-pair in hydrogen bonds that align with the long axis of the self-
b-sheets found in natural proteins.14 Ala, Ile, and Val exhibit the assembled nanofibers, typically taking on a b-sheet confirma-
highest tendency to self-pair; therefore, we incorporated these amino tion. This type of assembly results in a minimum at 220 nm and
acids into the peptide sequence. The EEE sequence was included to maximum at 190 nm in the CD spectrum.15 Peptide 3 exhibits a
provide the necessary hydrophilic/hydrophobic balance to achieve minimum at 199 nm, which most resembles a random coil
amphiphilicity. 4-Formylbenzoic acid (FBA) was added on resin confirmation (Fig. 2A). Above the CMC, however, a minimum
under the same conditions as typical amino acid couplings, giving at 220 nm is observed. Unfortunately, the high concentration
the modified peptide FBA-IAVE3 (2). Following cleavage and purifica- (10 mg mLÀ1) limited our ability to reliably collect data below
tion, peptide 2 was allowed to react with SATHA 1 in the presence of 205 nm, so the expected maximum at 190 nm could not be
TFA, employing molecular sieves as a drying agent. Full conversion observed. A similar spectrum is observed upon gelation at
to peptide 3 was observed after 20 min by 1H NMR spectroscopy, and 10 mg mLÀ1, indicating that a b-sheet structure likely forms
precipitation of the reaction mixture into CH2Cl2 afforded peptide 3, above the CMC and persists in the gel phase. Interestingly, a
which was purified by preparative HPLC.
negative peak at 313 nm increases in intensity upon aggrega-
Transmission electron microscopy (TEM) was used to character- tion and gelation, likely indicating that the SATO chromophore
ize the morphology of the self-assembled architecture of peptide 3 resides in a chiral environment above the CMC of the peptide.
(Fig. 1A). Conventional TEM images revealed long, one-
To further investigate the peptide secondary structure, IR
dimensional nanostructures with an average diameter of 10 nm. spectroscopy was used to analyze the amide I band region
In contrast, TEM of peptide 2 (Fig. S5, ESI†) revealed predominately (1600–1700 cmÀ1) of peptide 3. A strong absorption near
small spherical micelles around 10 nm in diameter and some 1630 cmÀ1 and a weaker absorption near 1680 cmÀ1 is typically
larger spherical structures around 50 nm in diameter. No one- observed when b-sheets are dominant.16 In contrast, absorp-
dimensional objects were observed.
tions for a-helices and random coils are both located near
Next, we examined whether peptide 3 was capable of form- 1650 cmÀ1. Below the CMC, an absorption peak centered at
ing a robust gel. Addition of aqueous CaCl2 solution to a 1 wt% 1637 cmÀ1 with no secondary absorption near 1680 cmÀ1 is
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Fig. 2 Characterization of peptide 3 at 0.25 mg mLÀ1 (red), 10 mg mLÀ1
(green) and 10 mg mLÀ1 in gel form (blue). (A) CD spectroscopy normalized
to largest peak. The solid horizontal line represents the 0 line; the vertical
dashed lines highlight the peaks indicative of random coil (199 nm) and
b-sheet (220 nm) structures. (B) IR spectroscopy with y-axes shifted for
clarity. The vertical dashed lines designate the amide I absorptions indi-
Fig. 4 Cytotoxicity of peptide 3 on endothelial cells in basal media with or
without Cys (1 mM). (A) In solution. (B) As 1 wt% aqueous gel. Viability was
measured after 24 h by live/dead staining (calcein AM and ethidium
homodimer).
cative of random coil (1637 cmÀ1) and b-sheet (1674 and 1620 cmÀ1
)
released from the gel for over 15 h (Fig. S8, ESI†). No H2S
release was observed in the absence of Cys.
conformations.
To assess whether peptide 3 could have potential use in vivo,
observed for peptide 3. Above the CMC the absorption shifts to a cytotoxicity study on mouse brain endothelial cells was
lower frequency at 1635 cmÀ1, but still without a secondary conducted. Cytotoxicity of peptide 3 in solution was evaluated
peak. Upon gelation, however, absorption peaks at 1620 cmÀ1 with and without Cys by measuring cell viability after 24 h
and 1674 cmÀ1 are present, indicative of a b-sheet structure.
(Fig. 4A). No toxicity was observed for the peptide 3 solution in
UV-vis and fluorescence spectroscopy data (Fig. S7, ESI†) either case up to 80 mM. Cytotoxicity of the peptide 3 gel at
show absorptions at around 320 nm and emissions with peaks 1 wt% was also measured with and without Cys (Fig. 4B). Again,
near 470 nm at all concentrations. No substantial redshift in no significant toxicity was observed.
fluorescence upon aggregation or gelation is observed. Taken
Lastly, H2S release from peptide 3 in the presence of
together, the spectroscopy data reveal that peptide 3 adopts a endothelial cells was analyzed using a turn-on fluorescent
random coil conformation below the CMC and increases in probe selective for H2S (DT-OH).17 The addition of peptide 3
b-sheet character above the CMC and upon gelation. Extensive and Cys to cells pre-incubated with DT-OH showed strong
aromatic stacking is not present in these aggregates but may fluorescence (Fig. 5). To confirm the fluorescence was a result
play a minor role in self-assembly.
of the H2S released from the peptide, several controls were
The rate of H2S release from peptide 3 was measured using performed: DT-OH only, DT-OH plus Cys, and DT-OH plus
an H2S-selective microelectrode (Fig. 3). This method allows for peptide 3 without Cys. Fluorescence was observed in neither
real-time monitoring of the concentration of H2S in solution.
Peptide 3 in solution released H2S more quickly than in gel
form with an earlier peaking time (91 vs. 110 min) and a higher
peak concentration (12.6 mM vs. 5.5 mM). Additionally, a period
of slow initial release was observed for the gel, in contrast to the
soluble peptide that immediately began releasing H2S upon
addition of Cys. We attribute this induction period and overall
slower release in gel form to limited diffusion of Cys into the
gel. Importantly, measureable concentrations of H2S were
Fig. 3 H2S release from peptide 3 (100 mM) in solution and as an aqueous
gel (total of 100 mM in peptide 3). Both experiments were run in PBS
containing 1 mM Cys.
Fig. 5 Phase, fluorescence, and overlay images showing strong fluores-
cence in endothelial cells preincubated with H2S probe DT-OH (5 mM) and
then treated with peptide 3 (100 mM) and Cys (500 mM). Scale bar = 50 mm.
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