Cross-Linking Approaches to Tuning the Mechanical Properties of

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Cross-linking approaches to tune the mechanical properties of peptide pi-electron hydrogels Wathsala Liyanage, Herdeline Ann M. Ardona, Hai-Quan Mao, and John D. Tovar Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00593 • Publication Date (Web): 11 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Bioconjugate Chemistry

Cross-linking Approaches to Tune the Mechanical Properties of Peptide π-electron Hydrogels

Wathsala Liyanage, † Herdeline Ann M. Ardoña, †‡ Hai-Quan Mao, ‡§ǁ and John D. Tovar*†‡§ †

Department of Chemistry, Krieger School of Arts and Sciences, Johns Hopkins

University, 3400 N. Charles St., Baltimore, MD 21218, USA ‡

Institute for NanoBioTechnology, Johns Hopkins University, 3400 N. Charles St.,

Baltimore, MD 21218, USA §

Department of Materials Science and Engineering, Whiting School of Engineering,

Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA. ǁ

Translational Tissue Engineering Center, Johns Hopkins School of Medicine,

Baltimore, MD 21287, USA.

*

E-mail: [email protected]

Web: http://sites.krieger.jhu.edu/tovar-group/ Tel: +1 410 5164358

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Abstract Self- assembling peptides are extensively exploited as bioactive materials in applications such as regenerative medicine and drug delivery, despite the fact that their relatively weak non-covalent interactions often render them susceptible to mechanical destruction and solvent erosion. Herein, we describe how covalent cross-linking enhances the mechanical stability of self-assembling πconjugated peptide hydrogels. We designed short peptide-chromophore-peptide sequences displaying different reactive functional groups that can form cross-links with appropriately modified bifunctional polyethylene glycol (PEG) based small guest molecules. These peptides self-assemble into one dimensional fibrillar networks in response to pH in the aqueous environment. The cross-linking reactions were promoted to create a secondary network locked in place by covalent bonds within the physically cross-linked (pre-assembled) π-conjugated peptide strands. Rheology measurements were used to evaluate the mechanical modifications of the network, and the chemical changes that accompany the crosslinking were further confirmed by infrared spectroscopy. Furthermore, we modified these cross-linkable π-conjugates by incorporating extracellular matrix (ECM) derived Ile-Lys-Val-Ala-Val (IKVAV) and Arg-GlyAsp (RGD) bioactive epitopes to support human neural stem/progenitor cell (hNSCs) differentiation. The hNSCs undergo differentiation into neurons on IKVAV derived π-conjugates while RGD containing peptides failed to support cell attachment. These findings provide significant insight into biochemical and electronic properties of π-conjugated peptide hydrogelators for creating artificial ECM to enable advanced tissue engineering applications.


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Introduction Self-assembled hydrogels derived from π-conjugated short peptides have been exploited in development of promising materials for optoelectronics as well as biomedical applications ranging from biosensors to tissue engineering.(1-5) The emergent biochemical and electronic properties of these peptide functionalized π-conjugates typically rely on extent of inter-molecular interactions and local geometries imposed on the π-electron moieties within the assemblies.(6-10) Peptide-π conjugates undergo spontaneous self-assembly into amyloid-like fibrils that primarily rely on weak non-covalent forces including Coulombic, hydrogen bonding and van der Waals interactions while the alignment of π-electron moieties within the assembly dictates the formation of electronically delocalized nanomaterials.(11-14) Hydrogelation is then induced by extended fibrillar entanglement or formation of fibril-fibril cross-links through additional noncovalent interactions.(15) Even though π-conjugated peptide based hydrogels have gained significant and growing attention as artificial materials to mimic the extracellular matrix (ECM), due to the transient and weak nature of the intermolecular interactions, the supramolecular assembly process often results in soft hydrogels with poor viscoelastic properties that fail to provide the necessary environment to provoke cell-specific functions.(16,

17)

It has been shown

that the mechanical properties of scaffolds and substrates can directly impact cell behavior, including cell motility, focal adhesion and cell growth. In addition, the mechanical properties of the microenvironment affect cell differentiation and the ability to uptake exogenous signaling molecules.(17) Current efforts to construct peptide based hydrogels for biological applications rely on mechanical modulation via chemical or enzymatic cross-linking. In particular, chemical crosslinking can be tuned to alter the mechanical properties of the soft hydrogels while enhancing the 3 ACS Paragon Plus Environment


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chemical diversity by providing an ability to functionalize with physical or biological cues. The mechanical properties of the resulting hydrogels depend on the degree of fiber branching and the type and strength of crosslinking between fibers.(18) Chemical cross-linking converts noncovalent interactions between peptides or their resulting nanomaterials into covalent bonds by connecting the reactive ends of specific functional moieties. However, gel formation relies on the extent of inter-molecular reactions while intra-molecular reactions may distract the gel formation. Recently, biocompatible “click chemistries” have been employed in cross-linking applications owning to their ease of use and efficient, highly selective reactions under mild cytocompatible conditions. The click chemistries include radical˗initiated thiol-ene(19) and thiolyne(20), Cu(I) catalyzed azide-alkyne cycloadditions (CuACC)

(21-23)

and strained˗promoted

azide-alkyne click cycloadditions (SPACC)(24-26) have been examined broadly for the formation of cross-links. Herein, we designed eight peptide-chromophore-peptide (7, 27) triblock peptides displaying specific functional moieties that can selectively react with appropriately modified small PEG based guest molecules to achieve network cross-linkings. Specifically, we examined the influence of several different cross-linking reactions (amine reactive cross-linking, azide-alkyne cycloaddition, thiol-ene reactions, enzymatic cross-linking) on the bulk rheological properties of the resulting hydrogels. These reactions can be triggered through a variety of external physical stimuli (e.g. sonication, photoexcitation). It was found that the coexistence of covalent and noncovalent interactions exert a strong influence on self-assembly/hydrogelation of these πconjugates while various side chain modifications on amino acids in the peptidic segments provide a convenient handle to tune the viscoelastic properties of resulting hydrogels. In addition, π-conjugates modified with laminin-derived Ile-Lys-Val-Ala-Val (IKVAV) and 4 ACS Paragon Plus Environment

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fibronectin Arg-Gly-Asp (RGD) were synthesized to impart specific biological activity. Human neural stem cells were seeded on hydrogels formed from RGD and IKVAV derivatized peptide

π−conjugates and the effects of these epitopes on neural differentiation were assessed. The IKVAV modified crosslinked π-conjugates showed an enhanced neuronal differentiation capability while RGD peptide hydrogels failed to support hNSCs. The cell studies on these cross-linked π-conjugates provide insights that will inform future rational design strategies for the construction of sophisticated optoelectronically relevant biomaterials. Results and Discussion Self-assembled peptide-based hydrogels are water-swollen three dimensional fibril networks cross-linked together with fibril-fibril entanglement, branching or chemical crosslinking. The supramolecular hydrogel derived from physical cross links or weak non covalent interactions are inherently dynamic in nature and susceptible to shearing by mechanical forces. Covalent crosslinks, on the other hand, are permeant junctions that can further modulate the resulting materials. Here, we illustrate the formation of novel π-conjugated peptide hydrogels using a two-component system, specifically functionalized peptides and PEG cross-linkers that enables hydrogel formation with tunable mechanics. PEG was chosen due to its biocompatibility, hydrophilicity and ease of modification with various functional groups, such as thiols, azides and amines. Design and synthesis. Herein, we have studied peptide-π-peptide triblock molecules that have pH dependent assembly, based on the well-established quaterthiophene (OT4) chromophore as the π-electron unit.(7, 27-29) The peptides were designed in line with our prior research such that the π-electron unit was flanked by two peptide sequences with the N˗ to ˗C peptide polarity



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extending outward from the π-electron unit. Unless stated otherwise, the peptide sequences here were designed to be soluble at basic pH (ca. pH 10) due to the electrostatic repulsion introduced by deprotonated aspartic acid (Asp, D) residues. At higher pH, these electrostatic repulsions frustrate the self-assembly while lowering the solution pH (ca. pH 2) screens these repulsions (by carboxylate neutralization) and promotes the assembly process via enabling intramolecular hydrogen bonding and other favorable enthalpic interactions. Hydrophobic and π−π interactions (30-32)

are also important determinants in self-assembly. Here, the relative contribution of π−π

interactions versus hydrophobic interactions was considered during the peptide designing. Valine (Val, V), a β-branched amino acid that has high β-sheet propensity was chosen as a prototypical hydrophobic sequence, and phenylalanine (Phe, F) was incorporated to also adopt the β-sheet secondary structure through hydrophobic and π−π interactions. The well-known selfassembling “Phe-Phe” motif was embedded within the peptide sequence to further improve the assembly propensity.(33, 34) The triblock peptides were manually synthesized using standard solid phase peptide synthesis (SPPS). The OT4 chromophore attachment was afforded by solid phase amidation and Pd-catalyzed conditions to initiate dimerization between quaterthiophene diacid and amine terminated resin bound peptides.(7) The peptides depicted in fig 1a were synthesized by incorporating Val and Phe to promote the assembly in aqueous solutions while reactive functional groups at the positions denoted with X provided locations for reactive cross-linking. We synthesized eight different peptide-OT4 conjugates of the general formula DXVVX-OT4XVVXD, and DXFFX-OT4-XFFXD, (where X =Lys (K), Propagyl Gly (Gp), Lys(Alloc) (K(Alloc)), and Gln (Q), all known participants in chemical/enzymatic cross-linking reactions).


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Figure 1. (a) Chemical structures of OT4-peptides (DXVVX and DXFFX) studied here; (b) Space-filling energy minimized model illustrating a possible assembly structure (top view) of DXFFX. The π-conjugated segment (quaterthiophene) is colored yellow. Self-assembly. The self-assembly of peptide-π−peptide triblocks under acidic and basic conditions was assessed in detail. Unless otherwise noted (e.g. for DKXXK peptides that assemble at basic pH), these triblock peptides exhibit molecularly dissolved states at basic pH (pH ≥8) while at lower pH (acidic pH), the peptides assembled into 1-D nanostructures. In the space-filling energy-minimized model (figure 1b) for DKFFK, the π-conjugated peptides are organized in an ideal orientation. This alignment facilitates a hydrogen-bond network between amide functionalities along the fibril axis and forces slightly slipped-cofacial interactions between π-units. The fibrils observed in hydrogels of these derivatives are likely to be composed of bundles of two or more units. The assembled π-conjugated peptides were monitored spectroscopically in dilute conditions (~10-6 M), using UV/Vis, PL and CD spectroscopy to probe the comparative electronic structure of the assembled materials. In the assembled state, the 7 ACS Paragon Plus Environment


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blue shift in the absorption and quenching of the photoluminescence is indicative of H-like aggregation of the chromophore subunits (Figure 2).(35) Representative photophysical characterization is shown in Figure 2 for DGpVVGp and DGPFFGP- peptides. The emission profiles depicted in Figure 2a,c show varied extents of quenching as a function of solution pH. CD spectra were also obtained for each peptide in monomeric and assembled state (Figure S15, Supporting information). Here, we analyzed the absorption of self-assembled materials in visible (550-400 nm) and UV region (both far UV (240-180 nm) and near UV (400-260 nm)). The CD spectrum of monomeric peptide state shows the extended baseline response, while assembled states show characteristic bisignate Cotton bands with a crossover (ellipticity is zero) near the absorption maximum of the peptides (Figure 2 and Figure S15, Supporting information). This exciton-coupled Cotton band found in assembled peptide solutions indicate the existence of OT4 chromophores in local chiral environments within each of the hydrogen-bonding network. The intensity and sign of Cotton effect varies dramatically due to the varying assembly propensities and extent of interactions between the chromophores, as we have shown previously. Nevertheless, the characteristic CD signals in the higher-energy amide-bond region suggest the formation of β-sheet like structures (minimum at 216 nm). Unlike other peptides, CD analysis of the DKVVK and DKFFK (Figure S15c and d, Supporting Information) at basic pH displayed characteristic Cotton bands with the inversion in the CD signal believed to originate from change in the supramolecular chirality of fibrils.


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Figure 2. Representative solution spectroscopy of DGpVVGp and DGpFFGP peptides (X = K): UV-vis and PL (λexc = 410 nm) of DGpVVGp (a) and DGpFFGp (c); CD spectra of DGpVVGp (b) and DGpFFGp (d) at basic pH (dashed curves) and acidic pH (solid curves). Unless otherwise noted, π-peptide conjugates for cross-linking reactions were dissolved in unbuffered water and mixed (vortexed) to obtain optically clear homogeneous solutions. These solutions exhibited pH values ~ 6-8 due to residual buffer from peptide purification. Bifunctional cross-linkers with identical reactive groups at either end of a spacer arm were used for this study. The peptide solutions were analyzed immediately after dissolution in unbuffered water by TEM and CD, and the data strongly suggest that the peptides are already assembled in unbuffered water (Figure S16a and b, Supporting information). However, the optically transparent 1 wt% solutions of these presumably assembled π-peptide conjugates in unbuffered water were unable to form self-supporting hydrogels (Figure S16c, Supporting information).



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Figure 3. (a) Model illustrating potential relative orientation of Lys side chains in the context of the self-assembled fibrils. (b) Amine reactive cross-linking agents. Amine cross-linking reactions. We examined simple amine based covalent crosslinking to enhance the viscoelasticity of supramolecular peptide hydrogels. N-hydroxysuccinimide (NHS) activated esters are widely applied in cross-linking approaches.(36) In general, NHS esters react with nucleophiles to release the NHS or sulfo-NHS group to create stable amide or imide bond between primary or secondary amines. Many NHS esters are insoluble in aqueous buffer and it is therefore necessary to dissolve them in organic solvents such as DMSO or DMF at high concentration so that only minimal volumes are delivered to the aqueous reaction medium. Instead,

we

employed

bis(sulfosuccinimidyl)glutarate

homobifunctional, (BS2G)(37)

with

water-soluble a

7.7

Å

sulfo-NHS spacer

arm,

ester and

A, B, 10

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bis(sulfosuccinimidyl)suberate (BS3)(37) with a 11.4 Å spacer arm, for cross-linking studies (Figure 3). Under physiological pH, these cross-linkers react with the free ε-amino groups of the lysine side chains to form stable amide bonds. We performed the crosslinking reactions in HEPES buffer at room temperature. The reactive DKVVK and DKFFK peptides were dissolved in HEPES to yield 1 wt% peptide solutions that were treated with A (10 mol%) or B (10 mol%) cross-linkers in HEPES at room temperature followed by gentle pipet mixing to afford crosslinked peptide materials (Table S4, Supporting information). The hydrolysis of the NHS ester competes

with

the

cross-linking

reaction

with

the

primary

amine.

The

self-

assembly/hydrogelation of cross-linked DKVVK and DKFFK peptides indicate some interesting trends (Table S4, Supporting information) in cross-linking reactions that are directly related to the spacer arm length of the cross-linkers, (A and B). In the simplified packing model for DKFFK, the peptide tape assemblies expose Lys residues to the solvent. This implies that these dangling residues can mediate inter and intra fibril covalent cross-links, determining the degree of fibril cross-links and hydrogel strength. The cross-linker A (with a shorter spacer arm length) seems to form better covalent bonds between peptide strands compared to cross-linker B (with a longer spacer arm length). The sequence hydrophobicity clearly influences the early molecular recognition event leading to fibril self-assembly, while amino acid variations induce morphological changes that dramatically influence the supramolecular behavior of these systems. The rheological strength of the hydrogels cross-linked with A was measured by an oscillatory frequency sweep with 0.2% strain (Figure 4). The storage modulus (G′) and loss modulus (G″) were determined for each cross-linked hydrogel in the linear viscoelastic region. The G′ and G″ of DKFFK were ~99000±11500 Pa and ~14000 ± 1700 Pa respectively, and the G′ and G″ of the DKVVK were ~31000±4000 Pa and 4000±500 Pa. Both hydrogels showed G′ and G″ values that



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were independent of frequency, consistent with rigid hydrogel formation. The observed difference in the rigidity of the gels formed by DKVVK vs DKFFK can be attributed to variable assembly propensity of peptides and their effective fibril cross-link density. These results suggest that complementary π−π interactions between aromatic side chains and functional group conversion play a role in assembly. The use of crosslinker B did not lead to the formation of selfsupporting gels, so rheological measurements were not recorded. We attempted to identify the cross-

linked products of DKVVK with linker A using electrospray ionization spectrometry (ESI) as a measure of cross-linking efficiency (Figure S17, Supporting information). According to the ESI fragmentation patterns, the cross-linked DKVVK displayed oligomer distributions extending to pentamers,

Figure 4. Strain sweeps (a, c) and oscillatory frequency sweeps (b, d) of DKVVK (a, b) and DKFFK (c, d) hydrogels following treatment with crosslinker A.


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Negatively stained TEM imaging was used to visualize the internal structure of resultant fibril morphologies. DKFFK formed rigid fibrils with A and B cross linkers (Figure S18, Supporting information). However, DKFFK showed distinct morphological changes with respect to the type of cross-linker. Crosslinking with A appears to form bundles of smaller fibrils with 5.1 ± 1.6 nm diameter (~consistent with a fibril on one peptide in width) while crosslinking with B appears to form wider fibrils with 6.5±1.8 nm diameter. DKVVK with cross-linker A shows the presence of extensive fibril bundles that are 8.0 ± 1.0 nm wide, however with cross-linker B does not appear to form any fibril assemblies (Figure S18, Supporting information).

It is

expected that the varying trend of fibril dimensions is due to changes in fibril-fibril cross-linking that depend on the spacer arm length of cross-linkers. Cu(I) catalyzed azide-alkyne cycloaddition cross-linking reactions (CuACC). CuAAC, commonly known as the “click reaction”, is a subclass of the classic Huisgen cycloaddition in which an azide reacts with an alkyne to form a triazole ring.(38, 39) This chemistry has found use in many complex biological systems without interfering with other chemical functionalities.(40, 41) In particular, the CuAAC reaction proceeds under physiologically relevant conditions (i.e. 5.5 ≤ pH ≤ 7.5 and T = 37 ± 2°C).(21) Herein, we have used a homobifunctional azido-modified PEG linker, 1-azido-2-[2-(2-azidoethoxy)ethoxy]ethane and suitably functionalized peptides, DGPVVGP and DGPFFGP where the alkynes were introduced via propargyl glycine residues (GP). Solutions of the alkyne containing peptides were prepared in unbuffered water (1 wt %) and treated with the azido-functionalized PEG cross-linker. The Cu(I) catalyst was generated in situ from copper(II) sulfate (1 mol %) using sodium ascorbate (10 mol %) as the reducing agent. Even though CuAAC is a reliable and fast reaction, the concentrations we used for these reactions were not sufficient to achieve the desired product or hydrogel formation as confirmed 13 ACS Paragon Plus Environment


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by FTIR. Inspired by the sonochemical Cu(I)-catalyzed synthesis of triazoles by Sreedhar,(42) we used ultrasound to accelerate the reaction at room temperature (Figure 5a). Interestingly, DGPFFGP formed a self-supporting hydrogel confirmed by vial inversion, visually indicating the formation of rigid hydrogel, while DGPVVGP stayed as a transparent un-invertible solution. Each mixture was then imaged by negatively stained TEM in order to reveal the distinct differences in morphology of self-assembled fibrils vs cross-linked fibrils. The fibrils that constitute the DGPFFGP had average diameters of 6 ± 1 nm, which decreased to 4 ± 1 nm upon cross-linking, indicating the efficiency of cross-linking reactions to afford more fibril-fibril entanglements (Figure 5b and c). DGPVVGP follows similar TEM trends (Figure S19), however, it fails to form a self-supporting hydrogel. These observations suggest that aromatic π−π interactions presented on the surface of the peptide tapes play a critical role in the self-assembly and subsequent fibrillization processes.


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Figure 5. (a) Schematic illustration of reaction between DGPFFGP homobifunctional azide crosslinker. TEM images of (b) 1 wt % DGpFFGp in unbuffered water (c) 1 wt% DGpFFGp under CuACC conditions. The rheological strength of the resulting DGPFFGP hydrogel was measured by oscillatory frequency sweep with 0.2% strain (Figure 6a). Rheology was collected in the linear viscoelastic region (Figure 6b). The DGPFFGP hydrogel has a G′ ~6000 ± 300 Pa G″ ~3700 ± 400 Pa that are independent of frequency. Even though the hydrogel formation (only observed for the DGPFFGP peptide) was observed upon short sonication, the CuACC reaction reaches a plateau in 30 min as confirmed by FT-IR spectroscopy (Figure 6c).

Figure 6. Oscillatory frequency sweep (a) and strain sweep (b) of DGpFFGp cross-linked hydrogel; (c) FT-IR spectra of homobifunctional azide cross-linker (green) and after to exposure to DGPFFGP after 30 min (red) and 60 min (blue). 15 ACS Paragon Plus Environment


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Thiol-ene reaction. Thiol-ene photoreactions have also been used for polymer based hydrogels, drug delivery and tissue engineering owing to their ease of use and the presence of thiols in many biomolecules. Herein, we exploited the thiol-ene reaction between alloxycarbonyl (alloc) functionalized peptides and thiol-terminated PEG molecules. The peptides DKaFFKa and DKaVVKa were synthesized using SPPS. The amine protecting group Alloc found in lysine (Ka) was used during the peptide synthesis and the commercially available homobifunctional 2,2′(ethylenedioxy)diethanethiol was used as the cross-linker. The Alloc-functionalized peptides were treated with the cross-linker (Figure 7a), and hydrogels were formed upon irradiation of the peptide-crosslinker solutions under cytocompatible doses of long wavelength ultraviolet (UV) light (10 Mw cm-2, 365 nm).


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Figure 7. (a) Visual depiction of the reaction between DKaVVKa with homobifunctional thiol cross-linkers leading to a self-supporting hydrogel. TEM images of (b) 1 wt% DKaVVKa (in unbuffered water) and (c) the hydrogel formed by treating 1 wt% DKaVVKa peptide with crosslinker. Under thiol-ene reaction conditions, the self-assembled fibrils of DKaVVKa in unbuffered water (Figure 7b) underwent distinct morphological changes over time. In unbuffered water the only observed morphology was the flat tape like structures, while under after treatment with the thiol-ene reaction conditions, DKaVVKa formed dense network of fibrils. TEM images of DKaVVKa peptide after the thiol-ene reaction clearly indicate the presence of ordered high aspect ratio bundled assemblies with diameters of 12±2 nm (Figure 7c). The rheological strength of resulting hydrogel after the UV curing process was assessed under dynamic frequency sweep (Figure 8a) experiments to measure the response of the materials to a constant strain applied with increasing frequency in the linear viscoelastic region (Figure 9b). The viscoelasticity of DKaVVKa hydrogel has a G′ of 6100 ± 900 Pa and G″ of 800 ± 200 Pa and independent of frequency less than 50 rads-1 consistent with a rigid hydrogel, while the products resulting from the cross-linking of DKaFFKa formed insoluble aggregates. FT-IR spectroscopy and rheology studies were used to monitor the UV curing of thiolene reactions between Alloc containing peptides and the thiol-terminated PEG cross-linker. The samples were irradiated outside the FT-IR spectrometer and then placed in the sample chamber. The FT-IR spectrum of the thiol-ene reaction was monitored after 10 min UV exposure time and compared with the peptide alone, the thiol-terminated PEG alone and peptide, thiol terminated PEG before UV curing, as shown in Figure 8c. At the beginning of the curing process, the thiol S-H stretch (at 2862 cm-1) is distinct. As the cross-linking progress, this peak begins to shrink in 17 ACS Paragon Plus Environment


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size as more of thiols react to form crosslinked networks. After 10 min, the thiol peak has decreased significantly (Figure 8c). Further, the PL spectra of thiol-ene solutions at the different time points were recorded to observe any changes in electronic structure with the UV curing (Figure 9d). Spectra were obtained at an excitation wavelength of 400 nm (excitation wavelength of OT4). The pre-assembled peptide DKaVVKa mixed with thiol terminated PEG linker fluoresces strongly when excited at 400 nm, but the PL quenches significantly during the UV curing process.

Figure 8. Oscillatory frequency sweep (a) and strain sweep (b) of DKaVVKa+PEG thiol crosslinked hydrogel;.(c) FT-IR traces of DKaVVKa thiol-ene reaction: DKaVVKa peptide (blue), DKaVVKa peptide+PEG dithiol after 10 min irradiation (red), PEG dithiol reagent (green), DKaVVKa peptide+PEG dithiol before irradiation (black dash line). (d) PL spectra of DKaVVKa with PEG dithiol 0-20 min after UV curing.


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Enzymatically cross-linked peptides. Enzymes are also routinely employed as catalysts for peptide cross-linking reactions. Transglutaminase (TG), a calcium-dependent enzyme, was used herein to catalyze a transamidation reaction between a primary amine (e.g. lysine) and a glutamine (Gln). The catalytic reaction promotes the glutamine deamination to form a peptideglutamyl-thioester at the active site of the enzyme.(43) Due to the inherent higher degree of substrate specificity within the TG, the enzymes only react with γ-amides of Gln residues leading to the formation of a thioester acyl-enzyme intermediate. Nucleophilic attack by a lysyl ε-amino group at the carbonyl moiety of the thioester intermediate generates a N-ε−(γ-glutamyl)lysine isopeptide bridge which is resistant to proteolysis. Gln containing peptide substrates DQFFQ and DQVVQ were synthesized as TG substrates. The 1 wt% peptide solutions in HEPES buffer (Ca2+ added) were treated with lyophilized guinea pig liver TG powder ( ≥1.5 units/mg per protein) and primary amine functionalized PEG linker, 2,2′-(ethylenedioxy)bis(ethylamine) and incubated at 37°C (Figure 9a).

Figure 9. (a) Schematic illustration of reaction between DQFFQ and a homobifunctional amine cross-linker and (b) TEM images of crosslinked 1 wt % DQFFQ. Oscillatory frequency sweep (c) and strain sweep (d) of DQFFQ TG crosslinked hydrogel. 19 ACS Paragon Plus Environment


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After short incubation times (1 h) at 37°C, crosslinked DQFFQ and DQVVQ selfassembled into rigid fibrils with mean diameters of 6 ± 1 nm and 5 ± 1 nm, respectively (Figure 9 a and Figure S20, Supporting information). In addition, these fibrils also underwent extensive bundling over time. The degree of fibril bundling could be due to effective crosslinking reactions between peptides and guest molecules. DQFFQ formed a hydrogel that was stable to vial inversion, while DQVVQ failed to form a self-supporting hydrogel under these conditions. Oscillatory rheology (Figure 9c, d) was used characterize the emergent viscoelastic properties of DQFFQ. The G′ of 4000 ± 600 Pa and G″ of 1500 ± 300 Pa of hydrogels were determined by a frequency sweep from 0 to 10 rad s-1 in the linear viscoelastic region at 0.2%.

Figure 10. Chemical structures of a) DGRKaVVKa b) VAVKIKaDDKa peptides.

π-Conjugated hydrogels as ECM mimetic scaffolds for neuronal cell culture. Selfassembling peptides are attractive ECM mimetics due to their excellent biocompatibility and biodegradability. The presentation of bioactive epitopes has an important influence in regulation of cell fate such as adhesion, migration and differentiation and these properties can be tailored by molecular design. Cell adhesion in the ECM relies on the formation of focal adhesion complexes between cell membrane integrins and ECM proteins.(44, 45) Therefore, the incorporation of cell 20 ACS Paragon Plus Environment

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adhesion sequences into self-assembled peptide nanostructures allows for the structural and functional mimicry of the ECM. However, the development of peptide based hydrogels as ECM mimetics has often been impeded by their poor mechanical characteristics. The mechanical cues presented by the ECM environment are crucial for regulating cell functions; in particular, the differentiation of stem cells towards a particular lineage can be directed by substrate and matrix mechanical properties.(46, 47) Softer gels (~100- 500 Pa) greatly favored neurons while harder gels (~1000-10,000 Pa) promoted glial cultures. In contrast, moduli of ~10 Pa inhibited the cell spreading and differentiation.(48) Herein, we synthesized Alloc containing peptide sequences with fibronectin-derived RGD and laminin-derived IKVAV peptide epitopes (Figure 10). The thiolene cross-linking conditions were employed with assembly, and the differences in the rigidity of the gels formed by cross-linked vs self-assembled VAVKIKaDDKa materials were assessed. The G′ and G″ values of cross-linked VAVKIKaDDKa were 349 ± 40 Pa and 41 ±7 Pa while selfassembled VAVKIKaDDKa were 92 ± 20 Pa and 31 ± 5 Pa respectively (Figure S23, Supporting information). Human neural stem cells (hNSCs) were seeded on both RGD (1 wt%) and IKVAV (1 wt%) containing peptide hydrogels. The DGRKaVVKa peptide (Figure 11a) hydrogel failed to support the cell attachment (Figure S24, Supporting information). However, hNSCs showed differentiation towards neuronal lineage after 5 days (without growth factors) exposure to the cross-linked IKVAV peptide as confirmed by the expression of β-tubulin observed from immunofluorescence staining. As for the hNSCs in cross-linked VAVKIKaDDKa (Figure 10b) at day 5, most of the cells were attached with significant neurite extension (Figure 11 panel 1), while in the self-assembled VAVKIKaDDKa (not cross-linked, Figure 11 panel 2) and on the tissue culture plate control there was significantly reduced neurite outgrowth (Figure 11 panel 3).



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Figure 11. In vitro cellular imaging study of hNSCs in laminin-derived IKVAV containing peptide hydrogel. Panel 1. Differentiation of hNSCs in crosslinked VAVKIKaDDKa gel, Panel 2. VAVKIKaDDKa before cross-linking reaction, Panel 3. Control experiment without gel, (a) 22 ACS Paragon Plus Environment

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hNSCs, (b) DAPI (nucleus staining) and (c) merged image (d) Quantification of neurite length (* n = 4, ** n = 3). We used a simple neurite tracer, an ImageJ plugin for automated tracing, to quantify the neurite outgrowth (Figure 11d) on VAVKIKaDDKa peptide before and after crosslinking reaction and on tissue culture plate (control). Human NSCs neurite lengths on the IKVAV based scaffold (382 ± 29 µm) were significantly longer than those on IKVAV before crosslinking reaction (108 ± 29 µm) and a tissue culture plate (53 ± 10 µm) after 5 days of culture. According to this cell based study the VAVKIKaDDKa cross-linked hydrogel supported cell attachment and differentiation by providing a suitable ECM mimic for hNSCs and also supported neurite outgrowth. Studies are underway to investigate how the different bioactive epitopes in the supramolecular hydrogel environment, along with varied optoelectronic stimuli, affect neural stem cell differentiation towards different lineages. Conclusion The study demonstrates the use of covalent-cross-linking strategies to tune the mechanical properties of optoelectronic peptide based hydrogels. Two general platforms were studies as substrates for cross-linking, one based on VV dipeptides (branched aliphatic) and one based on FF dipeptides (aromatic hydrophobe). Cross-linkable residues were placed flanking the VV and FF sequences.

We showed how a variety of covalent cross-linking chemistries could be

tolerated, from thiol-ene to CuAAC to enzymatic reactions. The emergent viscoelastic properties of the hydrogels that resulted from cross-linking reactions were quite variable. There was no clear trend observed between the rigidity of the hydrogels and the type of cross-linking reaction. Finally, the preliminary cell culture results for IKVAV containing thiol-ene click reactions



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provides significant design cues for the optimization of biological applications of this class of hydrogelators. The influence of hydrogel scaffolds on neuronal differentiation is of great interest for cell-based therapies. Future efforts will involve studies directed towards realizing advanced cell culture and tissue engineering applications. Associated Content Supporting Information The supporting information is available free of charge on the ACS Publication website. General experimental conditions and details on experimental procedures, the synthesis and characterization of peptides. Figures showing HPLC traces, ESI spectra, additional TEM images, Cell studies 1H NMR and 13C NMR. (PDF)

Acknowledgments We thank Johns Hopkins University and the National Science Foundation Biomaterials Program (DMR-1407493) for generous support. We also thank Prof. Jennifer H. Elisseeff for the use of G2 ARES (TA Instruments Inc.) rheometer for rheology experiments and the Center for Molecular Biophysics (JHU) for the use of circular dichroism spectrometer.


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Cross-Linking Approaches to Tuning the Mechanical Properties of

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