Tunable Pentapeptide Self‐Assembled β‐Sheet Hydrogels

Abstract Oligopeptide‐based supramolecular hydrogels hold promise in a range of applications. The gelation of these systems is hard to control, with minor alterations in the peptide sequence significantly influencing the self‐assembly process. We explored three pentapeptide sequences with different charge distributions and discovered that they formed robust, pH‐responsive hydrogels. By altering the concentration and charge distribution of the peptide sequence, the stiffness of the hydrogels could be tuned across two orders of magnitude (2–200 kPa). Also, through reassembly of the β‐sheet interactions the hydrogels could self‐heal and they demonstrated shear‐thin behavior. Using spectroscopic and cryo‐imaging techniques, we investigated the relationship between peptide sequence and molecular structure, and how these influence the mechanical properties of the hydrogel. These pentapeptide hydrogels with tunable morphology and mechanical properties have promise in tissue engineering, injectable delivery vectors, and 3D printing applications.

The self-assembly of oligopeptide sequences into nanostructures holds promise for ar ange of applications in biomedicine,food science,cosmetics,and nanotechnology. [1][2][3] These materials can be readily synthesized, providing hydrogel systems with robust mechanical properties. [3] Experimental and computational approaches have yielded aselection of di-and tripeptide sequences, [3][4][5][6][7] which have been proven to assemble into nanostructures and hydrogels under aqueous conditions,g enerating nanospheres, [8] fibrous and plate-like assemblies, [9,10] heterogeneous nanostructures, [4,11,12] and micelles and nanotubes. [13][14][15] To improve gelation characteristics,these small molecules often require either the inclusion of aromatic amino acid residues or as ynthetic terminal group. [1,[16][17][18][19] This introduces p-p stacking and hydrophobic interactions,w hich promote self-assembly and gelation. [3] However,s ynthetic terminal groups are not inherently biodegradable and are therefore less likely to be suitable for biological applications.A dditionally,m inor alterations in the sequence can significantly influence the selfassembly process,w hich makes both design and further functionalization difficult, whereby typical self-assembly rules cannot be applied. Thenative tripeptide sequences discovered to self-assemble into stable hydrogels have contained aromatic amino acids such as the KYF and DFY motifs. [3,20] Oligopeptides that consist of amino acids with aliphatic side chains have received less attention. [21,22] Furthermore,outside of tripeptide assemblies,t here have only been af ew studies which focused on oligopeptide sequences that are slightly extended in length (4-8 amino acids). In af ew cases,t hese studies have been based on short peptide fragments of larger polypeptides already known to self-assemble into nanostructures,s uch as NFGAIL [5,23] (fragment of human islet polypeptide) and KLVFFAE [24] (part of amyloid b [16][17][18][19][20][21][22]. Most recently,P appas et al. utilized ad ynamic combinatorial peptide library with dipeptide inputs and discovered that sequences of four residues (W4, F2L2) and six residues (F6, L6) formed higher-order assemblies.A dditionally,t he eight-residue FDFSFDFS sequence was also able to form aself-supporting hydrogel. [22] We hypothesized that exploring the self-assembly of pentapeptides would provide flexibility in chemical design and gelation propensity,w hile allowing for simplicity in synthesis for future applications.W er eport three pentapeptide sequences that are free of aromatic groups and can form highly robust hydrogels with stiffnesses spanning two orders of magnitude from 2t o2 00 kPa ( Figure 1). The peptide sequences discovered were found to contain three aliphatic isoleucine (Ile) residues,a na mino acid with ah igh propensity to form b-sheets. [25,26] These aliphatic amino acids were further combined with two aspartic acid (Asp) residues, which improve the solubility of hydrophobic Ile.T hen, upon protonation, charge recognition/hydrogen bonding drives b-sheet self-assembly and hydrogel formation. To further investigate the self-assembly of the pentapeptide sequences, the positions of the charged Asp residues were systematically altered to generate three different charge distributions ( Figure 1): Asp flanking ac entral Ile region (DI3D), Asp at the N-terminus of the sequence (D2I3), and Asp alternating with Ile residues (IDIDI). Using these three sequences and their different architectures,weaimed to explore the relationship between amino acid sequence and molecular structure, and their influence on the mechanical properties of the hydrogel.
In an initial screen, we tested different peptide designs and sequence lengths,w hich yielded differences in solubility and gelation. These included an additional pentapeptide sequence (DI4), at etrapeptide (DI2D), and av aline variant (DV3D). TheDI4 sequence was not soluble in aqueous media and could not be purified. TheD I2D and DV3D sequences could be solubilized in aqueous media, but no obvious selfassembly or gel formation was witnessed. From this initial screen, ar atio of 2Asp to 3Ile within ap entapeptide sequence proved most successful, enabling both purification of the peptides and subsequent assembly into robust hydrogels.
Peptide stock solutions were dissolved at 1and 2wt% in ab asic aqueous media at pH 10 through sonication. These stock solutions were then aliquoted onto ah ydrophobic surface and asmall volume of HCl pipetted onto each droplet to achieve an eutral pH. Upon the addition of HCl, the peptide solution gelled and could be manipulated with tweezers ( Figure 1).
Them echanical properties of the hydrogels were studied by oscillatory shear rheology.H ydrogel formation was verified, as the storage modulus (G')e xceeded the loss modulus (G'')atboth 1and 2wt% (see Figures S2 and S3 in the Supporting Information). Thefrequency sweeps show that the mechanical properties of all the hydrogels were independent of oscillation frequency,and this is consistent across the three sequences studied (see Figures S2 Aand S3 A). The hydrogels were also evaluated under the application of shear strain. Themoduli remained in the linear elastic region up to strains of around 1% with little change in G',f ollowed by as ignificant decrease in G' for strains exceeding 2% (see Thes tiffness of the hydrogels was dependent on both hydrogel concentration and the charge distribution of the peptide sequence (Figure 2A). At both 1a nd 2wt%,t he D2I3 sequences generated the stiffest gels,a nd under the same conditions the IDIDI hydrogels exhibited the lowest G' values.Comparing IDIDI (1 wt %) and D2I3 (2 wt %) hydrogels,the G' value increased by two orders of magnitude from 2 to 200 kPa, respectively.T hese stiffness values are in the region of many soft tissues and compare well to previously published peptide hydrogel systems,i ncluding aromatic peptides [4,17] and peptide-amphiphile hydrogels. [27,28] The ability to tune the G' value across al arge range holds great promise for applications in tissue engineering,g iven that the behavior of cells has been found to be heavily influenced by the mechanical properties of their surrounding environment. [29,30] One of the primary benefits of using noncovalent interactions is their ability to reform after deformation, allowing self-assembled hydrogels to recover their mechanical properties after the application of high strains. [31,32] To investigate the self-healing performance of these systems, as eries of step strain measurements were carried out ( Figure 2B). All the hydrogels displayed as teep incline in modulus,recovering around 50 %ofG' within 5min, followed by ap lateau and complete recovery between 10 and 20 min. These self-healing properties can also be cycled (see Figure S4). Theability to repeatedly recover mechanical properties highlights the dynamic nature of these hydrogels,inwhich the b-sheets can adopt more energetically favorable and mechanically robust conformations over time. [32] Thedynamic nature of these systems is further supported by their shear-  thinning characteristics,w hich were evaluated using flow sweeps (see Figure S5 A,B). All the hydrogels displayed typical shear-thinning behavior with viscosity decreasing linearly with increasing shear stress.T he combination of both self-healing and shear-thinning capabilities renders these hydrogels ideal for biomedical applications that require recovery after significant deformation, such as injectable therapies or 3D printing.
To investigate the relationship between supramolecular structure and mechanical properties,t he secondary structure of the peptide assemblies in the hydrogels were studied using spectroscopic techniques.T he CD spectra of the hydrogels resembled a b-sheet, with am inimum between 220 and 230 nm ( Figure 3A;s ee also Figures S5 Aa nd S6 A). This structure was supported by the amide Ir egion of FTIR spectra (see Figures S6 Ba nd S7 B), in which all hydrogels displayed aprominant peak at 1630 cm À1 indicating a b-sheet conformation. [33,34] However,differences in the CD and FTIR spectra were evident for each of the sequences studied. The CD spectra differed in intensity and were red-shifted relative to those of model b-sheets,w hich typically have am aximum at 195 nm and am inimum at 216 nm. [34] TheCDsignatures of b-sheets are known to have greater variability than those of other peptide secondary structures. [28] b-Sheets have both significant intermolecular and intrastrand hydrogen bonding. [35] Furthermore,p eptides can form anti-parallel, parallel, or mixed b-sheets,which will influence both the strands in the assemblies as well as the networks they form. [28] We analyzed the relative red-shifts in the CD minima of the different hydrogels.T he IDIDI sequence provided the softest gels and had the smallest red-shift at both 1and 2wt% ( Figure 3A,B). In contrast, DI3D and D2I3 materials had similar red-shifts with no significant difference in G' at 1wt%.H owever,a t2wt %t he D2I3 sequence was significantly stiffer and had the greatest red-shift in the CD spectra at this concentration ( Figure 3A,B). Previous studies have suggested that ar ed-shift in the CD spectra of b-sheets is representative of more twisted and distorted arrangements. [28,36,37] Thed egree of twisting of b-sheets is centered around the middle of the sequence. [34] In twisted b-sheets,the hydrogen-bonding distance increases as the angle between two peptides increases,w eakening the intermolecular forces and hydrogen bonds on the periphery of the b-sheet. [38,39] This will influence the intermolecular forces between individual peptide sequences in the b-sheet and the morphology of the structures present in the hydrogel. [35] Ad ifference in b-sheet peak intensity at 220-230 nm was also observed. TheC D measurements were performed at the concentration found in the hydrogel, and in some cases the hydrogels were partially opaque,w hich is likely to result in some fraction of the light being scattered, influencing peak intensity.
Them orphology of the different hydrogels was characterized by cryo-focused ion beam scanning electron microscopy (cryo-FIB SEM). In this technique,h ydrogel samples are plunged into liquid ethane,r apidly freezing the water content to obtain at hin layer of vitreous ice.T his preserves the morphology of the structure in aqueous solution and eliminates drying effects that can be generated when using other preparation techniques.Afocused ion beam (FIB) of gallium ions is then used to mill across-section in the sample with an exposed featureless face.R aising the temperature of the stage to 100 8 8Ccauses water to slowly sublime away from this face,r evealing the underlying physical structure (see Figure S10). This technique allows for imaging of the hydrogels in their native state and in the presence of bound water, overcoming major artefacts associated with drying and water removal (more details of this technique can be found in the Supporting Information). [40] From the electron micrographs collected, it is evident that the charge distribution in the peptide sequence influences the microstructures of the hydrogels (Figures 4; see also Figures S8 and S9). TheIDIDI hydrogels are comprised of highaspect-ratio nanofibers,w hich at 2wt% are several microns in length, extending to the height of the trench milled by the FIB (Figure 4A). At al ower concentration (1 wt %), the IDIDI hydrogels still maintain the same nanofibrillar morphology,b ut the fibers are shorter in length ( Figure 4B). In comparison, both the D2I3 and DI3D sequences have more entangled microstructures.T he D2I3 materials are formed from platelike assemblies interconnected by some fibrous domains ( Figure 4C,D);t hese observations were further supported by cryo-transmission electron microscopy images of the D2I3 hydrogels at 2wt% (see Figure S11). Similarly, the DI3D hydrogels are comprised of some nanofibers but mostly contain dense regions of fibrous bundles (Fig-Figure 3. A) Circular dichroism of the pentapeptide hydrogels at 1a nd 2wt%.The minimum between 220 and 230 nm is typically indicative of b-sheet formation. B) b-Sheet red-shift from 216 nm taken from the circular dichroism spectra. ure 4E,F). In summary,i tc an be observed that the more entangled structures have agreater degree of interconnectivity between the assemblies.
Recently,i th as been reported that Asp positioning can influence the stacking orientation of tripeptide b-sheet assemblies. [20] Shifting the Asp from the C-to the N-terminus was shown to invert the conformation from ap arallel to an antiparallel b-sheet. [20] Similarly,b oth the D2I3 and DI3D peptides contain charged Asp species situated at the termini of the sequence with regions of three repeat Ile residues. Previous studies on polyisoleucines reported that sequential Ile-rich structures are more stable in twisted parallel b-sheet arrangements. [35,41] TheFTIR spectra for the D2I3 and DI3D sequences had two minor peaks at 1655 and 1675 cm À1 (see Figures S6 Ba nd S7 B). It has been shown that twisted b-sheets (both in parallel and antiparallel conformations) can display an amide Isplitting with apeak between 1680 and 1690 cm À1 as well as ap eak at 1650 cm À1 . [28,34] Although the D2I3 and DI3D sequences in this study cannot be explicitly defined as being in an antiparallel or ap arallel orientation, these observations are in agreement with the red-shifted CD spectra found, which suggests that both the D2I3 and DI3D hydrogels contain more twisted b-sheets.
Thet erminal charged groups coupled with weakened hydrogen bonds on the periphery of the D2I3 and DI3D b-strands will result in ag reater potential to form ionic interactions and further hydrogen bonds with other neighboring strands.T hese interactions will give rise to the entangled and interconnected assemblies attributed to the D2I3 and DI3D hydrogels (Figures 4C-F;s ee also Figure S11). In the IDIDI sequence,t he Asp residues are positioned more centrally with singular b-sheet-forming amino acids (Ile) in the middle and at the termini. Given that this arrangement does not contain as eries of repeat Ile residues,i ti sl ikely to provide less twisted b-sheets.T hese types of structure will have less entropy and disorder,with hydrogen bonds between sequences being equal in length across the peptide chain, which is likely to facilitate planar stacking arrangements and result in the high-aspect-ratio nanofiber assemblies in Figure 4A,B.
Thedifferent types of intermolecular interactions and the high-order assemblies they form influence the mechanical properties of the pentapeptide hydrogels systems.L arger platelike domains that are more interconnected/entangled provided the stiffest hydrogels,whereas the high-aspect-ratio fibers in the IDIDI hydrogels behave like discrete structures with little entanglement between neighboring fibers,resulting in softer hydrogels.Furthermore,the 1wt% IDIDI hydrogels with shorter fiber lengths have less surface area for entanglement, which corresponded with an order-of-magnitude decrease in G' from 60 to 2kPa. These three different peptide designs demonstrate that alteration of the position of the b-sheet-forming amino acids and charge distribution of the sequence serves as au nique approach to control the morphology and tune the mechanical properties of the resultant hydrogel. Both substrate stiffness and substrate shape have been shown to influence cellular behavior. [29,30,42] Therefore,w ith control over both of these parameters,t he hydrogels have potential to act as tissue-engineering scaffolds and matrices.
We have reported three pentapeptide sequences free of aromatic groups,w hich can form robust hydrogels with gelation induced through changes in pH. We demonstrated that the stiffness of the hydrogels can be tuned across two orders of magnitude (2-200 kPa) by altering the concentration and charge distribution of the peptide sequence.F ormed through noncovalent interactions,the hydrogels showed selfhealing and shear-thinning behavior through reassembly of the physical cross-links.T oe xplore the relationship between molecular design and the mechanical properties of the resulting hydrogel, we utilized spectroscopic techniques, which verified the b-sheet structure.D epending on the peptide sequence and its charge distribution, different degrees of red-shift were evident in the CD spectra, which corresponded to the different morphologies of the selfassembled structures within the hydrogels.C ryo-FIB SEM indicated that the IDIDI hydrogels were formed from highaspect-ratio nanofibers.I nc ontrast, the D2I3 and DI3D hydrogels had more entangled and interconnected structures, resulting in the stiffest hydrogels.T hese pentapeptide selfassembled hydrogels with tunable morphology and mechanical properties,a sw ell as self-healing and shear-thinning characteristics,p rovide ap romising platform for tissue engineering,i njectable delivery vectors,a nd 3D printing applications.