Modulating Supramolecular Peptide Hydrogel Viscoelasticity Using Biomolecular Recognition

Article abstract of DOI:10.1021/acs.biomac.7b00925

Self-assembled peptide-based hydrogels are emerging materials that have been exploited for wound healing, drug delivery, tissue engineering, and other applications. In comparison to synthetic polymer hydrogels, supramolecular peptide-based gels have advantages in biocompatibility, biodegradability, and ease of synthesis and modification. Modification of the emergent viscoelasticity of peptide hydrogels in a stimulus responsive fashion is a longstanding goal in the development of next-generation materials. In an effort to selectively modulate hydrogel viscoelasticity, we report herein a method to enhance the elasticity of β-sheet peptide hydrogels using specific molecular recognition events between functionalized hydrogel fibrils and biomolecules. Two distinct biomolecular recognition strategies are demonstrated: oligonucleotide Watson-Crick duplex formation between peptide nucleic acid (PNA) modified fibrils with a bridging oligonucleotide and protein-ligand recognition between mannose modified fibrils with concanavalin A. These methods to modulate hydrogel elasticity should be broadly adaptable in the context of these materials to a wide variety of molecular recognition partners.

Full text of DOI:10.1021/acs.biomac.7b00925

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Article
Modulating Supramolecular Peptide Hydrogel
Viscoelasticity Using Biomolecular Recognition
John T. M. DiMaio, Todd M. Doran, Derek M. Ryan, Danielle M. Raymond, and Bradley L. Nilsson
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00925 • Publication Date (Web): 05 Sep 2017
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Modulating Supramolecular Peptide Hydrogel Viscoelasticity Using
Biomolecular Recognition
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John T. M. DiMaio, Todd M. Doran,# Derek M. Ryan, Danielle M. Raymond, and Bradley L.
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Nilsson*
University of Rochester, Department of Chemistry, Rochester, NY 14627
Email: bradley.nilsson@rochester.edu; Phone: 585ꢀ276ꢀ3053
#Current address: Department of Medicinal Chemistry, University of Minnesota Twin Cities, 308
Harvard Street SE, Minneapolis, MN 55455
Abstract
Selfꢀassembled peptideꢀbased hydrogels are emerging materials that have been exploited
for wound healing, drug delivery, tissue engineering, and other applications. In comparison to
synthetic polymer hydrogels, supramolecular peptideꢀbased gels have advantages in
biocompatibility, biodegradability, and ease of synthesis and modification. Modification of the
emergent viscoelasticity of peptide hydrogels in a stimulus responsive fashion is a longstanding
goal in the development of nextꢀgeneration materials. In an effort to selectively modulate
hydrogel viscoelasticity, we report herein a method to enhance the elasticity of βꢀsheet peptide
hydrogels using specific molecular recognition events between functionalized hydrogel fibrils
and biomolecules. Two distinct biomolecular recognition strategies are demonstrated:
oligonucleotide WatsonꢀCrick duplex formation between peptide nucleic acid (PNA) modified
fibrils with a bridging oligonucleotide and proteinꢀligand recognition between mannose modified
fibrils with concanavalin A. These methods to modulate hydrogel elasticity should be broadly
adaptable in the context of these materials to a wide variety of molecular recognition partners.
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Introduction
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Hydrogels are emerging materials for biomedical applications that include tissue
engineering1, 2, wound healing3ꢀ5, drug delivery3, 6, 7, and immunotherapy.8 Selfꢀassembled
peptide based hydrogels are an attractive alternative to traditional polymerꢀbased gels for
applications requiring inherently softer materials due to advantages in biocompatibility and
biodegradability.9ꢀ19 In addition, spontaneous selfꢀassembly of peptide nanofibril networks from
relatively simple peptide sequences provides opportunities for modifying the emergent hydrogel
properties through alteration of the peptide sequence.20ꢀ23
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The design of selfꢀassembled hydrogels with varying emergent viscoelasticity that is
correlated to peptide sequence is a longꢀstanding goal for applications that involve interaction of
the gel network with cells. The ability to tune hydrogel viscoelasticity can influence the utility of
the gels for interactions with different cell types5, 24 and can potentially influence other physical
characteristics, such as rate of release for drug delivery.7, 25ꢀ27 The viscoelasticity of noncovalent
peptide gels relies primarily on the concentration and entanglement efficiency of the fibril
network. This limits, to some extent, the ability to modify elasticity of the resulting hydrogels
once the network has been established. Strategies to selectively tune the viscoelasticity of selfꢀ
assembled peptide hydrogels, including covalent crossꢀlinking of assembled peptide fibrils and
alteration of environmental stimuli, are primarily useful to change the elasticity as the network
initially forms.28ꢀ30 There are few examples of modification of hydrogel elasticity for
supramolecular peptide hydrogels after network formation.31ꢀ33
Herein we describe the stimulusꢀresponsive enhancement of selfꢀassembled peptide
hydrogel viscoelasticity by specific noncovalent crossꢀlinking of peptide nanofibrils using a
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biomolecular recognition strategy. The amphipathic Acꢀ(FKFE)2ꢀNH2 peptide is a frequently
studied example of selfꢀassembling peptide that rapidly forms βꢀsheet bilayer nanofibrils at low
ꢀM concentrations; hydrogel networks of these nanofibrils are established at concentrations
exceeding ~4 mM.9, 20, 34ꢀ40 At lower concentrations, the fibrils that assemble do not form a
sufficiently entangled network to elicit gelation of aqueous solvents. We hypothesized that
hydrogels of Acꢀ(FKFE)2ꢀNH2 nanofibrils could be rigidified by selective crossꢀlinking of the
fibrils via noncovalent interactions between biomolecular recognition motifs appended to the
selfꢀassembled nanofibrils and molecules that bind to these motifs (Figure 1). This specific crossꢀ
linking would rigidify the hydrogel by selectively reinforcing the fibril network. This strategy
has previously been applied to gelation of synthetic polymer gels.41ꢀ43 Two strategies were
explored to test this hypothesis: (1) crossꢀlinking of peptide fibrils with appended
oligonucleotide mimetics by complementary bridging DNA strands and (2) crossꢀlinking of
saccharideꢀmodified peptide fibrils by lectins with multiple recognition sites for the nanofibrilꢀ
appended carbohydrate. In both cases the viscoelasticity of peptide nanofibril hydrogels was
enhanced upon introduction of the bridging biomolecules. This work demonstrates fundamental
design principles for in situ stimulusꢀresponsive modification of the emergent properties of
noncovalent hydrogels.
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Figure 1. Pictorial representation of hydrogel viscoelasticity enhancement through bridging of
biomolecules appended to supramolecular peptide fibrils via specific interactions. These
interactions reinforce the crossꢀlinking density of the hydrogel network, thus increasing the
elasticity of the bulk hydrogel.
Materials and Methods
Peptide Synthesis. Cꢀterminal amide peptides were synthesized on Rink amide resin
(100–200 mesh, 0.2 mmol gꢀ1 loading, Advanced ChemTech, Louisville, KY), and loaded
manually
using
fluorenylmethoxycarbonyl
(Fmoc)
chemistry
and
standard
hydroxybenzotriazole/N,N,N,NꢀtetramethylꢀOꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)uranium
hexafluorophosphate (HOBt/HBTU) protocols. Peptides were synthesized using standard Fmoc
protection with HBTU/HOBt activation on a microwaveꢀequipped CEM Liberty Peptide
Synthesizer (CEM, Matthews, NC). Nꢀterminal acetylation was performed (where specified)
with 20% acetic anhydride in dimethylformamide (DMF) for 40 min. Peptides were cleaved
from the solid support using trifluoroacetic acid (TFA) cleavage cocktail, consisting of TFA,
triisopropyl silane (TIS), and water (95:2.5:2.5, v/v/v) for 1 h at room temperature. The
supernatant was collected, concentrated, and precipitated from cold diethyl ether (ꢀ78 ºC); the
precipitated peptide was collected by centrifugation. The organic supernatant was decanted and
the pellet was dissolved in dimethylsulfoxide (DMSO) for purification. Purification and
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characterization data are provided in the Supporting Information (Tables S1 and S2, Figures S1–
S8).
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Peptide nucleic acid (PNA) synthesis. PNA sequences were synthesized using similar
Fmoc deprotection methods. PNA sequences at the Cꢀterminus of peptides were coupled
iteratively onto Rink amide resin (100–200 mesh, 0.05 mmol/gꢀ1 loading, Advanced ChemTech,
Louisville, KY) preloaded with lysine. Fmoc deprotection was performed in 20% piperidine in
DMF for 5 min. The resin was washed three times with DMF, and then three times with CH2Cl2.
Couplings were then performed twice for 30 min using diisopropylcarbodiimide/1ꢀhydroxyꢀ7ꢀ
azabenzotriazole (DIC/HOAt) activation. Nꢀterminal PNA sequences were prepared similarly,
but following the initial synthesis of the full (FKFE)2 sequence. Acetylation was performed with
20% acetic anhydride in DMF for 40 min at room temperature. Peptide/PNA hybrids were
cleaved from the solid support using trifluoroacetic acid (TFA) cleavage cocktail, consisting of
TFA, triisopropyl silane (TIS), and water (95:2.5:2.5, v/v/v) for 1 h at room temperature. The
supernatant was collected and concentrated. Peptides were precipitated from cold diethyl ether (ꢀ
78 ºC) and collected by centrifugation. The organic supernatant was decanted and the pellet was
dissolved in DMSO for high performance liquid chromatography (HPLC) purification.
Attachment of 2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
2H-pyran-2-yl)oxy)acetic acid to (FKFE)2 peptides. The synthesis of the deprotected
mannosyl carboxylic acid, 2ꢀ(((2S,3S,4S,5S,6R)ꢀ3,4,5ꢀtrihydroxyꢀ6ꢀ(hydroxymethyl)tetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)acetic acid, was carried out as described in the literature.44 This deprotected
mannosyl carboxylic acid (118 mg, 500 µmol) was activated with 1ꢀ
[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate
(HATU) (190 mg, 500 µmol) in CH2Cl2/DMF (1 : 1, v/v) (5 mL). This mixture was allowed to
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stand for 10 min. The solution was the added to Nꢀterminally deprotected (FKFE)2 on the solid
support. This mixture was allowed to stir for 6 h. The peptide on resin was washed, dried, and
then cleaved using the procedure described supra.
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HPLC Purification of Peptides and Hybrid PNA or Mannose Peptides. Peptides were
purified on a Shimadzu LCꢀ6AD high performance liquid chromatograph (HPLC) (Shimadzu,
Columbia, MD) equipped with a reverseꢀphase (RP) C18 column (BEH300, 10 µm, 19 x 250
mm, Waters, Milford, MA). A gradient of acetonitrile (0.1% TFA) and water (0.1%) at 10 mL
minꢀ1 was used to separate products. Eluent was monitored by UV absorbance at 215 and 254
nm. Purity was assessed by injection on an analytical Shimadzu LCꢀ10ATVP HPLC instrument
equipped with a RPꢀC18 column (Waters, Milford, MA). Peptides were characterized via
MALDIꢀTOF analysis. Pure peptides were frozen and lyophilized to a powder for later use. See
Supporting Information (Tables S1ꢀS2 and Figures S1ꢀS8 for purification and characterization
data for all peptides).
Peptide Self-Assembly. Lyophilized peptides were dissolved in 6:4 mixtures of water
and acetonitrile (v/v). Concentration was determined via correlation to an HPLC calibration
curve. The appropriate volumes of peptides were mixed (as appropriate) and lyophilized. The
lyophilized material was then dissolved in unbuffered water at appropriate concentrations and
vortexed for 1 min.
Circular dichroism (CD) Analysis of Nanofibrils. Peptides were prepared as outlined
above at a concentration of 500 µM in unbuffered water. CD spectra were then recorded on an
AVIV 202 CD spectrometer (AVIV Biomedical, Lakewood, NJ). Spectra were obtained with a
0.1 mm quartz cuvette (Hellma, Concord, ON, CA) at 25 ºC from 260 to 190 nm with a 3 s
average collecting time per step at a 1.0 nm step and 1.0 nm bandwidth. Background subtraction
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was performed, followed by conversion to molar ellipticity. The resulting spectra were then
smoothed using a least squares fit on the AVIV software.
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Negative-Stain Transmission Electron Spectroscopy (TEM). Peptides were prepared
as outlined above at a total peptide concentration of 500 µM. 10 µL of the assembled peptide
solution was applied to a 200ꢀmesh carbonꢀcoated copper grid. After standing for 1 min, excess
fluid was removed by capillary action. 10 µL of filtered 5% uranyl acetate was placed on the grid
and allowed to stand for 1 min. After standing, the uranyl acetate was removed by capillary
action. Grids were then allowed to air dry for 5–15 min. TEM micrographs were obtained using
an Hitachi 7650 transmission electron microscope with an accelerating voltage of 80 kV. Fibril
dimensions determined using ImageJ (NIH, rsbweb.nih.gov/ij/).
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PPH/Ac-(FKFE)2-NH2 Hybrid Gel Formation. PNAꢀbearing fibril hydrogels were
prepared as outlined above at a total peptide concentration of 4.2 mM (3 mM Acꢀ(FKFE)2ꢀNH2,
0.6 mM PPH1, and 0.6 mM PPH2). The mixed peptides were vortexed for 1 min and allowed to
stand for 1 day. The resulting gel was analyzed on an ARꢀG2 rheometer (TA Instruments).
Hydrogels in the presence of bridging DNA were prepared as outlined above, except 100
nmol of DNA was added after dissolution of the peptides. Almost immediately upon addition of
DNA, the solution visibly increased in viscosity. This material was vortexed for 1 min and
allowed to stand for 1 day. The resulting gel was analyzed on an ARꢀG2 rheometer (TA
Instruments).
Man-(FKFE)2 Hydrogel Formation. Manꢀ(FKFE)2ꢀNH2 hydrogels were prepared as
described above at a concentration of 3.5 mM peptide in unbuffered water. To test for
conconavalin A (ConA) mediated crossꢀlinking, 1 µL of ConA (5 g/L) was added to select gels
([ConA] = 2.4 µM). These hydrogels were assessed by oscillatory rheology.
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Rheological Analysis of Gels. Rheology was performed on an ARꢀG2 rheometer (TA
Instruments) operating in oscillatory mode, using a 20 mm parallel plate geometry. Samples
were prepared as outlined above and applied directly to the rheometer stage. Following gel
application, a dynamic frequency sweep was performed for 0.1–40 rad sꢀ1 with 0.2% strain at 25
ºC (this was found to be within the linear viscoelastic region for the gels as determined by a prior
strain sweep experiment). All reported values for G´ and G˝ are the average of at least three runs.
Data shown is representative data.
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Results & Discussion
Herein, we report a strategy to modify the viscoelasticity of selfꢀassembled peptide
hydrogels in situ by exploiting specific molecular recognition phenomena to noncovalently
crossꢀlink, and thereby rigidify, the hydrogel network. Our first approach to accomplish this
objective exploits WatsonꢀCrick baseꢀpair formation between olignonucleotide mimetics
appended to Acꢀ(FKFE)2ꢀNH2 nanofibrils and a complementary oligonucleotide strand that acts
as an interfibril bridge (Figure 2). For these studies, peptide nucleic acid (PNA) oligomers were
exploited (Figure 3). PNA is a polyamide oligonucleotide mimetic that retains the ability to bind
to complementary oligonucleotides (including DNA, RNA, and other PNA strands).45, 46 PNA is
conveniently appended to peptides since they are synthesized using nearly identical solidꢀphase
methods.47ꢀ50 PNA has been demonstrated to form duplexes with both DNA and RNA with high
affinity (the average free energy of binding (ꢁGº) is ꢀ6.5 ± 0.3 kJ molꢀ1 bpꢀ1)51 and specificity.52,
53
The neutral, uncharged backbone of PNA also allows sequenceꢀselective duplexes to be
formed even at low ionic strength, enabling the use of PNA in unbuffered aqueous solutions
without added salts.48, 53
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Figure 2. Conceptual illustration of WatsonꢀCrick mediated fibril crossꢀlinking of peptide
nucleic acid (PNA) bearing selfꢀassembled Acꢀ(FKFE)2ꢀNH2 nanofibrils (PPH1/PPH2
nanofibrils) with a bridging oligonucleotide that is complementary to the PPH1 and PPH2 PNA
sequences. The bridging oligonucleotide forms selective noncovalent crossꢀlinks between fibrils,
which strengthens the hydrogel network thereby increasing the emergent hydrogel
viscoelasticity.
Figure 3. Chemical structure of the peptide nucleic acid (PNA) aminoethyl glycine backbone
and the appended nucleobases.
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This strategy was explored using the peptide, PNAꢀpeptide conjugate, and DNA
oligonucleotide sequences shown in Table 1. Acꢀ(FKFE)2ꢀNH2 was used as the fundamental selfꢀ
assembling peptide motif that constitutes the assembled peptide nanofibrils. The PPH1 PNAꢀ
peptide chimera has a PNA decamer appended to the Nꢀterminus of an (FKFE)2ꢀNH2 peptide,
while the PPH2 chimera has a PNA decamer attached to the Cꢀterminus of Acꢀ(FKFE)2. The
PNA oligomers on PPH1 and PPH2 are nonꢀcomplementary to each other. A bridging DNA
oligonucleotide that is complementary to the PPH1 PNA oligomer at the 5' end and to the PPH2
PNA oligomer at the 3' end was designed. These DNA segments are connected via a linker
sequence that has no complementarity to either PNA strand. Conceptually, PPH1 and PPH2 can
be coassembled with Acꢀ(FKFE)2ꢀNH2 at varying ratios to provide nanofibrils that display the
PNA sequences at a density that is proportional to the coassembly ratio of PPH1:PPH2:Acꢀ
(FKFE)2ꢀNH2. When these PPH1/PPH2 hybrid nanofibrils are mixed in the presence of the
bridging DNA oligonucleotide, the fibrils will be crossꢀlinked via WatsonꢀCrick duplex
formation, theoretically increasing the elasticity of the hydrogel network (Figure 2).
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Table 1. Sequences of peptide, peptide/PNA hybrid, and DNA biopolymers. For peptides, upper
case letters are amino acids and lower case letters are PNA residues; PEG is 8ꢀaminoꢀ3,6ꢀ
dioxaoctonoic acid. For the DNA sequence, the underlined nucleotides are complementary to the
underlined PNA residues in PPH1 and the bold faced nucleotides are complementary to the bold
faced residues in PPH2.
Biopolymer
(FKFE)2
PPH1
PPH2
Bridging
DNA
Sequence
AcꢀFKFEFKFEꢀNH2
AcꢀttctctctgaꢀPEGꢀFKFEFKFEꢀNH2
AcꢀFKFEFKFEꢀPEGꢀtttctaatgtꢀKꢀNH2
5´ꢀ TCAGAGAGAATACCTCAACATTAGAAA ꢀ 3´
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The requisite peptides were prepared and the assembly properties of PPH1 and PPH2
coassembled with Acꢀ(FKFE)2ꢀNH2 were characterized. The peptides were synthesized by
standard Fmoc solidꢀphase methods. It was found that appending PNA analogs onto selfꢀ
assembling (FKFE)2 peptides did not impede assembly of these peptides into nanofibrils. All
nanofibrils described in this section were prepared in unbuffered water. Acꢀ(FKFE)2ꢀNH2, PPH1,
and PPH2 were mixed in a 5:1:1 ratio (500 ꢀM total peptide) and the assemblies formed from
this mixture were compared to assemblies of Acꢀ(FKFE)2ꢀNH2 alone. Circular dichroism (CD)
analysis of Acꢀ(FKFE)2ꢀNH2 yielded the expected the minima at 218 and 205 nm, as have
previously been observed for fibrils of this peptide. These minima correspond to characteristic βꢀ
sheet secondary structure (218 nm) and πꢀπ effects between Phe side chain groups in the
hydrophobic core (205 nm) (Figure 4A). The CD spectrum of PPH1/PPH2/Acꢀ(FKFE)2ꢀNH2
hybrid nanofibrils was dominated by strong absorbance at 220 nm by the PNA nucleobases,
complicating assessment of secondary structure of these PNAꢀbearing fibrils. However, TEM
images of the selfꢀassembled Acꢀ(FKFE)2ꢀNH2 and the coassembled PPH1/PPH2/Acꢀ(FKFE)2ꢀ
NH2 hybrid fibrils indicated the formation of similar nanotape fibril assemblies (Figure 4B and
4C). Fibrils formed from just Acꢀ(FKFE)2ꢀNH2 were tapeꢀlike in appearance and 8.4 ± 0.4 nm in
width, consistent with previous reports;54ꢀ56 the PPH1/PPH2/Acꢀ(FKFE)2ꢀNH2 hybrid fibrils
were a mixture of tapes and helical ribbons 8.1 ± 0.7 nm wide. Helical ribbons are also observed
at earlier incubation times with Acꢀ(FKFE)2ꢀNH2 fibrils, but these evolve into nonꢀhelical
structures in time.39, 57 While the attachment of PNA does not appear to impede assembly (even
PPH1 and PPH2 alone are capable of selfꢀassembly without mixing with Acꢀ(FKFE)2ꢀNH2, see
Supporting Information, Figures S9 and S10), the evolution from initial helical morphologies to
nonꢀhelical tapes is slightly slowed.
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Figure 4. CD spectra and TEM micrographs of Acꢀ(FKFE)2ꢀNH2 (black) and a combination of
Acꢀ(FKFE)2ꢀNH2, PPH1, and PPH2 (red). All concentrations are 500 µM (total peptide
concentration). The combination of Acꢀ(FKFE)2ꢀNH2, PPH1, and PPH2 is a total peptide
concentration of 500 µM, but in a ratio of 5 : 1 : 1 (Acꢀ(FKFE)2ꢀNH2 : PPH1 : PPH2). (A) CD
spectra; (B) TEM Micrograph of Acꢀ(FKFE)2ꢀNH2; and (C) TEM micrograph of a combination
of Acꢀ(FKFE)2ꢀNH2, PPH1, and PPH2.
Next, the viscoelasticity of hydrogels composed of either Acꢀ(FKFE)2ꢀNH2 fibrils or
PPH1/PPH2/Acꢀ(FKFE)2ꢀNH2 hybrid fibrils was characterized. These hydrogels were prepared
in unbuffered water. Oscillatory rheology was used to measure viscoelasticity as a function of
the storage modulus (G´) and the loss modulus (G˝) of the respective materials. Gels were
formed in each case at peptide concentrations of 4.2 mM. Dynamic frequency sweeps from 0–40
rad sꢀ1 at 0.2% strain (which falls in the linear viscoelastic region for these gels as determined by
previous strain sweep experiments) were performed to measure the storage and loss moduli of
these materials. Acꢀ(FKFE)2ꢀNH2 hydrogels and PPH1/PPH2/Acꢀ(FKFE)2ꢀNH2 hybrid hydrogels
without any bridging oligonucleotide had similar rheological characteristics, with G´ values of
148.2 ± 10.4 Pa and a G˝ values of 20.7 ± 9.17 Pa (Figure 5). Consistent with previous
experiments, these gels are soft materials.9, 10, 37
The gels formed in the presence of a bridging DNA oligonucleotide (added at a
concentration of 0.6 mM, approximately equimolar to each appended PNA oligomer), exhibited
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increased G´ values of 245 ± 11.3 Pa and G˝ values of 23.08 ± 3.37 Pa. Again, the DNAꢀbridged
hydrogels were also prepared in unbuffered water since salts are not required for the formation of
duplexes between uncharged PNA oligomers with negatively charged DNA. The addition of
DNA doubled the storage modulus of the resulting gels while leaving the loss modulus
unchanged, indicating a significant rigidification of the hydrogel in the presence of a specific
biomolecular recognition motif that forms fibrilꢀfibril crossꢀlinks. While the bulk rheological
properties of the hydrogel materials indicate enhancement of the crossꢀlinked entangled state,
extensive TEM imaging of the network failed to provide quantifiable measurements of the
microscopic degree of additional crossꢀlink formation. The addition of nonꢀcomplementary DNA
oligonucleotides resulted in no change of the observed storage and loss moduli. In addition,
modifying the ratio of PNA displayed on the fibrils or altering the concentration of bridging
DNA did not alter the degree of enhancement of elasticity in the gels. These experiments confirm
that noncovalent crossꢀlinking of selfꢀassembled peptide nanofibrils with specific biomolecular
recognition motifs can be used to modify the emergent viscoelasticity of the resulting hydrogels.
Oligonucleotide crossꢀlinking of the nanofibril network is an effective strategy to rigidify the
network in situ.
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Figure 5. Dynamic frequency sweep of Acꢀ(FKFE)2ꢀNH2, PPH1, and PPH2 in the absence of
DNA (red) and presence of bridging DNA (black). The combination of Acꢀ(FKFE)2ꢀNH2, PPH1,
and PPH2 is a total peptide concentration of 4.2 mM in a ratio of 5 : 1 : 1 (Acꢀ(FKFE)2ꢀNH2 :
PPH1 : PPH2).
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The observation that modifying the ratio of PNA displayed on the fibrils or altering the
concentration of bridging DNA has no significant effect on the elasticity in the resulting gels
warrants some discussion. The biophysics of hydrogelation in this system is complicated and the
degree of G' enhancement does not have a clean linear relationship with PNA concentration (or
DNA concentration) because the network is influenced not only by PNA/DNA crossꢀlinking of
the fibrils, but also by the inherent entanglement of the fibrils that occurs at gelation
concentrations. As such, there is no convenient method to estimate what ratio of the appended
PNA is 1) available for interaction with crossꢀlinking DNA and 2) is actually participating in a
DNA bridged crossꢀlink. It is possible that some of the appended PNA is inserted into the
hydrophobic bilayer of the peptide nanofibrils and are thus unavailable to form DNA crossꢀlinks.
It is also possible that the formation of DNAꢀbridged interfibril crosslinks perturbs the
equilibrium between the assembled and disassembled states of the peptide nanofibrils by
mechanically pulling the PNAꢀbearing peptides from the assembled nanofibrils. The data shows
that the addition of bridging DNA does enhance hydrogel viscoelasticity in these materials,
which does not, however, preclude the existence of these complicated states.
Efforts to elicit solꢀgel transformations of the PNAꢀdecorated nanofibrils were also
disappointing. We dissolved the peptide nanofibrils at concentrations below 4 mM where no selfꢀ
supporting hydrogels were formed and attempted to trigger gelation by addition of the bridging
DNA. While the addition of DNA visually increased the viscosity of these solutions, we were
unable to find conditions where selfꢀsupporting hydrogel formation was triggered. These
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observations reinforce the fundamental importance of nanofibril concentration, in addition to
specific interfibril crossꢀlinking interactions, in the formation of entangled hydrogel networks of
selfꢀassembled peptide materials. Despite these limitations, the data presented herein clearly
indicates that specific noncovalent crossꢀlinking interactions between the selfꢀassembled fibrils
can significantly alter the emergent viscoelasticity of these hydrogel materials.
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A second approach to affect specific fibrilꢀfibril crossꢀlinking of Acꢀ(FKFE)2ꢀNH2
hydrogels was explored that exploits lectin recognition of carbohydrates appended to the fibril
network. Concanavalin A (ConA) is a lectin protein that binds to mannose ligands (Ka of 6.8 ×
103 Mꢀ1 for monovalent ConA,58 7.54 × 103 Mꢀ1 for divalent ConA,58 8.2 × 103 Mꢀ1 for
tetrameric ConA).59 At neutral pH, ConA is a homotetramer (Figure 6B), while at pH 3–4 it is
dimeric. Thus, ConA in an oligomeric form is capable of binding multiple mannose molecules.
We hypothesized that ConA would therefore be an effective crossꢀlinking agent for (FKFE)2
fibrils that bear mannose. Accordingly, we prepared the Manꢀ(FKFE)2ꢀNH2 peptide shown in
Figure 6A to test this hypothesis.60, 61
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Figure 6. Structures of the mannoseꢀbearing peptides and tetrameric concanavalin A (ConA).
(A) Structure of the mannoseꢀbearing (FKFE)2 peptide (Manꢀ(FKFE)2ꢀNH2). (B) Structure of
tetrameric ConA (PDB entry 1QDC).62
Manꢀ(FKFE)2ꢀNH2 effectively selfꢀassembled into
βꢀsheet nanofibrils in unbuffered
water similarly to the parent Acꢀ(FKFE)2ꢀNH2 peptide. As shown in Figure 7A, the CD spectrum
of Manꢀ(FKFE)2ꢀNH2 fibrils is identical to that of Acꢀ(FKFE)2ꢀNH2 (Figure 4A), with
characteristic minima at 205 nm and 218 nm. TEM images also indicate that Manꢀ(FKFE)2ꢀNH2
readily selfꢀassembled into nanofibrils in both the absence and presence of ConA (Figure 7B and
7C). These fibrils are 3.5 ± 0.6 nm in diameter, which is narrower than fibrils derived from Acꢀ
(FKFE)2ꢀNH2 alone. These narrower dimensions are likely due to a steric effect of the Nꢀ
terminal mannose, which likely precludes lateral βꢀsheet interactions that result in the wider
fibrils observed for Acꢀ(FKFE)2ꢀNH2.
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Figure 7. CD spectrum and TEM micrographs of Manꢀ(FKFE)2ꢀNH2. (A) CD spectrum of Manꢀ
(FKFE)2ꢀNH2; (B) TEM micrograph of Manꢀ(FKFE)2ꢀNH2 fibrils without ConA; and (C) TEM
micrograph of Manꢀ(FKFE)2ꢀNH2 fibrils with ConA.
Oscillatory rheology confirms that ConA effectively crossꢀlinks the Manꢀ(FKFE)2ꢀNH2
nanofibril network (Figure 8). Hydrogels were again formed at 4.2 mM concentrations of Manꢀ
(FKFE)2ꢀNH2. These gels are formed in unbuffered water, with the pH of the gels measured at
5.6. This slightly acidic gel pH is presumably due to residual TFA from the purification of Manꢀ
(FKFE)2ꢀNH2. At this pH, ConA will likely be a mixture of dimers and tetramers. Without the
addition of ConA, the resulting gels had an average G´ values of 148.3 ± 8.5 Pa and G˝ values of
14.9 ± 10.53 Pa, similar to Acꢀ(FKFE)2ꢀNH2 gels at this same concentration. Upon the addition
of ConA (2.4 µM), the resulting gels exhibited G´ values of 466.3 ± 60.8 Pa and G˝ values of
43.7 ± 5.2 Pa. This represents a significant enhancement of hydrogel elasticity affected by
selective biomolecular crossꢀlinking of the fibril network. Interestingly, the storage modulus
displays slight nonꢀlinearity, with G´ increasing at higher frequencies in the measurement. This
may be due to enhancement in ConA crossꢀlinking at higher frequencies as a function of the
relatively low binding affinity of ConA to mannose. This effect may also be due to perturbation
of the multimeric state of ConA (dimer versus tetramer) as the rheological experiment
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progresses. The addition of ConA to unmodified (nonꢀmannose bearing) Acꢀ(FKFE)2ꢀNH2 fibrils
had no effect on hydrogel viscoelasticity.
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Figure 8. Dynamic frequency sweep of Manꢀ(FKFE)2ꢀNH2 hydrogels (4.2 mM) in the absence
of ConA (red) and presence of ConA (black).
As with the DNAꢀbridged system, the data presented from the lectinꢀbridged hydrogels
raises some additional questions regarding the materials. It is not clear how the formation of
ConAꢀbridged interfibril crosslinks influences the stability of the peptide nanofibrils. The
possibility of mechanically disrupting the mannoseꢀbearing peptides from the assembled
nanofibrils exists and the data available does not clearly address the extent to which this may
occur and what the consequences may be. In addition, the binding affinity of ConA to mannose
is relatively weak. While a similar degree of enhancement is observed in the DNAꢀbridged and
ConAꢀbridged hydrogels, ConA was added at a much higher concentration (2.4 ꢀM) compared
to the DNA bridge (100 nM) at a constant peptide concentration of ~4 mM. Thus, the lower
binding affinity of ConA for its mannose ligand has a significant impact on the sensitivity of the
emergent hydrogel properties to the presence of the crossꢀlinking agent. Thus, the design of these
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types of materials for biological applications must carefully consider ligand binding affinity and
the available concentration of the ligand in the environment in which the hydrogel is to be
employed.
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These studies further confirm that bridging biomolecules can effectively crossꢀlink
peptide nanofibril hydrogels that display appropriate ligands for the biomolecule and that this
strategy is effective for the in situ rigidification of the hydrogel network. The ConA/mannose
strategy demonstrates that protein/ligand interactions are an effective crossꢀlinking method for
proteins that are multimeric and can bind multiple equivalents of the ligand. The ConA and
oligonucleotide strategies also show some limitations of this strategy in terms of the degree of
enhancement of hydrogel viscoelasticity. First, the enhancements observed in this study were
significant, but fairly modest. Hydrogel formation of selfꢀassembled peptide nanofibrils is highly
concentration dependent. In the case of Acꢀ(FKFE)2ꢀNH2, selfꢀassembly occurs at ꢀM
concentrations, but hydrogelation of these selfꢀassembled nanofibrils requires concentrations
greater than ~ 4 mM. Attempts to elicit sol to gel state transitions by crossꢀlinking with DNA or
ConA at peptide concentrations below 4 mM where the peptides were selfꢀassembled but not
hydrogels were only modestly successful. Visually, these solutions became more viscous, but
failed to meet the standard of selfꢀsupporting hydrogels by the inversion test. For these types of
peptide hydrogels, selfꢀsupporting gel formation depends highly on peptide concentration,
meaning this biomolecular crossꢀlinking strategy is most valuable for in situ modification of
hydrogel viscoelasticity and not for triggering gelation a priori. A second limitation that should
be appreciated is that not all crossꢀlinking events will be interfibril based on this design strategy.
Intrafibril crossꢀlinks, which may not contribute as strongly to enhancement of hydrogel
viscoelasticity, are also undoubtedly formed. Finally, the longꢀterm stability of these gels will
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undoubtedly influence the applications for which this strategy will be useful. Future studies will
focus on the longꢀterm stability of the crossꢀlinked hydrogels as a function of ligand binding
affinity and time.
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Despite these limitations, biomolecular crossꢀlinking of ligands that are displayed on
peptide nanofibrils in a multivalent fashion is an effective and simple strategy to modify
hydrogel viscoelasticity in situ. The ease with which biomolecular ligands can be displayed on
selfꢀassembled peptide fibrils makes this a convenient strategy to enhance hydrogel rigidity that
should be readily adaptable to a wide variety of biomolecule/ligand systems. This could be
highly attractive for hydrogels in biological environments in which proteins or other
biomolecules of interest are present. For example, overexpression of a protein of interest in cells
in tissue culture experiments could result in in situ enhancement of hydrogel viscoelasticity,
which could influence cell fate or other properties of the gel, such as rate of delivery of small
molecule drugs packaged within the gel. This strategy could also be readily adapted to sensing
applications. We anticipate future studies in which these types of applications are explored using
biomolecular recognition to modify emergent properties of selfꢀassembled peptide hydrogels.
Conclusion
We have demonstrated that the emergent properties of hydrogels derived from selfꢀ
assembled amphipathic peptides can be modified by crossꢀlinking the fibril network via
noncovalent bimolecular recognition phenomena. Specifically, Acꢀ(FKFE)2ꢀNH2 nanofibril
hydrogels have been rigidified using two noncovalent crossꢀlinking strategies: (1)
oligonucleotideꢀmediated crossꢀlinking by complementary WatsonꢀCrick base pair formation and
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(2) lectinꢀmediated crossꢀlinking of fibrils displaying mannose. These results are significant in
that they establish proof of principle for the use of noncovalent crossꢀlinking to modify the
viscoelasticity of peptide hydrogels, soft materials for which the elastic properties are typically
limited by the concentration of the fibril network. In addition, the noncovalent crossꢀlink
strategies are specific and selective, indicating that these or similar molecular recognition events
can be used to strategically alter hydrogel rigidity in situ in response to biological
microenvironments. This design strategy can be readily employed to tune the properties of selfꢀ
assembled peptide hydrogels as a function of environment for a wide array of applications,
including drug delivery and wound healing.
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Supporting Information
Purification and characterization data for all peptides and additional CD spectra and TEM
micrographs are provided in the Supporting Information.
Acknowledgements
We acknowledge support from the National Science Foundation (DMRꢀ1148836) for this
research. We also thank Karen Bentley (URMC Electron Microscope Research Core) for
assistance with TEM experiments.
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Figure 1. Pictorial representation of hydrogel viscoelasticity enhancement through bridging of biomolecules
appended to supramolecular peptide fibrils via specific interactions.
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Figure 2. Conceptual illustration of Watson-Crick mediated fibril cross-linking of peptide nucleic acid (PNA)
bearing self-assembled Ac-(FKFE)2-NH2 nanofibrils (PPH1/PPH2 nanofibrils) with a bridging oligonucleotide
that is complementary to the PPH1 and PPH2 PNA sequences. The bridging oligonucleotide forms selective
noncovalent cross-links between fibrils, which strengthens the hydrogel network thereby increasing the
emergent hydrogel viscoelasticity.
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Figure 3. Chemical structure of the peptide nucleic acid (PNA) aminoethyl glycine backbone and the
appended nucleobases.
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Figure 4. CD spectra and TEM micrographs of Acꢀ(FKFE)2ꢀNH2 (black) and a combination of Acꢀ(FKFE)2ꢀNH2,
PPH1, and PPH2 (red). All concentrations are 500 µM (total peptide concentration). The combination of Acꢀ
(FKFE)2ꢀNH2, PPH1, and PPH2 is a total peptide concentration of 500 µM, but in a ratio of 5 : 1 : 1 (Acꢀ
(FKFE)2ꢀNH2 : PPH1 : PPH2). (A) CD spectra; (B) TEM Micrograph of Acꢀ(FKFE)2ꢀNH2; and (C) TEM
micrograph of a combination of Acꢀ(FKFE)2ꢀNH2, PPH1, and PPH2.
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