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Chapter 8
The Design and Applications of Beta-Hairpin Peptide Hydrogels Peter Worthington1 and Darrin Pochan*,2 1Department
of Biomedical Engineering, University of Delaware, Newark, Delaware 19716, United States 2Department of Material Science and Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716, United States *E-mail:
[email protected].
This chapter reviews the history, development, and applications of the beta-hairpin peptide family of hydrogelators developed by the Schneider and Pochan laboratories. Peptide design provides for molecules that intramolecularly fold with consequent intermolecular assembly into a tightly defined fibrillar network in desired solution conditions. The resultant hydrogel network is described as an “injectable solid” due to the innate property of being able to flow under shear and immediately reheal back into a solid network on the cessation of shear. The ability to modify the peptide sequence confers a high degree of tunablility over the resultant hydrogel network properties and the solution conditions in which a gel is formed. This tunability leads to integration of the hydrogel within many applications such as drug delivery and complex cell culture.
Introduction Supramolecular gels are an important subclass of hydrogels formed from molecular self-assembly, both of high and low molecular weight compounds, that rely on non-covalent interactions to generate the highly hydrated, fibrous networks with desired material properties (1–3). The resulting supramolecular, physical (as opposed to covalent) networks tend to be easily modified due to the relative simplicity of their molecular building blocks and the ease with which © 2018 American Chemical Society Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
changes can be made in the molecular self-assembly pathway. With respect to changes in the molecular assembling molecules, slight alterations in chemical structure can result in targeted changes to the hydrogel nanostructure that have large implications in the resulting bulk material. With respect to alteration of the molecular self-assembly pathway, supramolecular hydrogel networks are highly dependent on their solution conditions; temperature, pH, and salt concentration can be used to trigger and either expedite or slow self-assembly in a number of ways. This combination of molecule and assembly pathway design results in tunable materials with a high degree of customization available. Biomolecules are particularly capable of functioning as the basic building block of a supramolecular gel, including DNA, RNA, proteins, synthetic polypeptides/peptides, and carbohydrates. While each group has its own advantages and disadvantages, amino acid-based molecules are of particular interest due to their natural ability to form higher order structures and potential cyto- and biocompatibility. Alpha-helical and beta-sheet secondary structures result in decidedly regular local structure. Hydrophobic collapse, hydrogen bonding, and electrostatic interactions can drive specific self-assembly from peptide backbone and side chain interactions. Put together, the above attributes allow proteins, polypeptides, and peptides to be designed in a biomimetic fashion for specific, desired self-assembly mechanisms. Peptides are a particularly useful class of amino acid-based hydrogelators due to their comparatively small size, ease of design and synthesis, and ease of solution processing. The ability to design peptide intra- and intermolecular interactions from the bottom up results affords a thorough understanding of the effect of each amino acid within a molecule and assembly pathway and the implications from changing an individual residue. There is a rich history of using peptide based hydrogels taking advantage of their propensity to self-assemble using secondary structure, hydrophobic collapse, and electrostatic interaction. Adams (4, 5), Gazit (6, 7), and Ulijn (8, 9) have taken advantage of the aromatic interaction of Fmoc-peptides to generate hydrogels. Collier (10, 11), Conticello (11), Hartgerink (12, 13), and Woolfson (5, 14) have used the alpha-helix secondary structure to great effect while Boden (15), Saiani (16, 17), and Zhang (18, 19) have relied on the beta-sheet as a building block for hydrogel formation. The beta-hairpin is a specific type of beta-sheet interaction that relies on the interaction of two covalently connected beta-sheet strands. This type of molecular structure is the result of a de novo designed peptide placing two amphiphilic amino acid strands with high beta-sheet propensity flanking a central turn sequence. The solution conditions can be altered to trigger the intramolecular folding of the molecule into a beta-hairpin conformation. Importantly for supramolecular assembly and hydrogel formation, only after the monomer folds does intermolecular assembly of the fibrillar network begin. The dependence of the intermolecular assembly on the intramolecular folding provides a high degree of control over hydrogelation, only occurring in desired solution conditions and with desired kinetics. This chapter will focus on the design and modification of the MAX family of beta-hairpin peptide hydrogels designed by Schneider and Pochan, the work within both the Schneider and/or Pochan laboratories to understand the basic self-assembly mechanisms and material properties, and resulting applications explored. 140 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Structure The MAX family of peptides are designed around the motif of two locally amphiphilic arms flanking a central turn sequence. MAX1, the original peptide design is a 20 amino acid sequence ((VK)4-VDPPT-(KV)4-NH2) with the two amphiphilic arms being 4 sets of alternating valine and lysine around the central type II’ turn structure of valine, d-proline, proline, and threonine (20). The research concerning this class of peptides has spanned a number of small and large modifications to the amino acid sequence, both in the arms and the turn sequences, with large implications for the supramolecular assembly pathway, the nanostructures formed, and the hydrogel material properties and applications, all of which will be discussed herein. (Figure 1)
Figure 1. TEM of self-assembled nanofibrillar structures and the proposed self-assembled structure of the fibrils and dimension. Reprinted in part with permission from Phys. Rev. Lett. 2004, 93 (26 I), 1–4. Copyright 2004 American Physical Society (20). 141 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Folding Mechanism The MAX1 amino acid sequence in pure pH 7 water exists in solution in the random coil confirmation. When the proper folding conditions (temperature, pH, salt concentration) are met the peptide is able to fold into its hairpin conformation. The charge state of the arm lysines is critical for controlling when this change occurs. The charges keep the arms from folding together into a parallel beta sheet. If the pH of the solution is increased the lysines can be deprotonated, if the salt concentration in the solution is increased the charges can be screened, and if the temperature of the solution increases the rate of hydrophobic collapse also increases (21–23). These three variables can be used together or independently tuned to control beta-hairpin folding and consequent hydrogelation. After the hairpin is formed it is stabilized by hydrogen bonding between the two arms of the peptide. The formation of the individual hairpins results in the beginning of the supramolecular assembly. (Figure 2) The hairpin is facially amphiphilic with a hydrophobic valine face and a hydrophilic lysine face. Two hairpins will stack burying the hydrophobic valine face between them. More hairpin pairs will assemble laterally continuing to bury the hydrophobic valines and stabilized by intermolecular hydrogen bonding. This process results in the formation of physically stabilized nanofibrils with a valine core sandwiched between two lysine faces. Recently, the molecular packing of the beta-hairpins within the fibrils has been well characterized. The two layers of hairpins within individual nanofibrils always have their turn sequences on opposite sides from each other (24). Also, the vertical hairpin pairs do not stack perfectly over one another but offset by half the width of a hairpin.(Figure 3) The hydrogel network forms as entanglement interactions between different fibrils occur and defects during the fibril self-assembly process result in nucleation points for a new fibril branch. An example of gelation occurs in phosphate buffered saline at pH 7.4 at 37 C with 150 mM NaCl-the assembly process of 0.75 wt% MAX1 takes approximately 30 minutes and results in a hydrogel with a stiffness of approximately 1 kPa. Modifications in the procedure and/or molecule that results in a faster gelation process (high concentration of peptide, higher salt concentration, higher temperature, alteration of the peptide design) would result in a stiffer overall hydrogel.
Material Properties Like most hydrogels MAX is a highly hydrated network, typically approximately < 97% water by weight. MAX is also a peptide based material, typically made from solid phase peptide synthesis. This results in a material that is highly tunable and cyto-. Biocompatible (26). Because of the self assembling nature of the hydrogel it is simple to encapsulate a number of different things inside the hydrogel, primarily cells or therapeutic compounds. The stiffness of typical hydrogels made from the assembly of beta-hairpins is between 10-10,000 Pa as measured by oscillatory rheology. The most exciting material property is its shear thinning behavior; after the hydrogel has formed into a solid, a shear force can be applied to the hydrogel resulting in the hydrogel flowing like a liquid 142 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
(27). As soon as the force is stopped the hydrogel immediately reheals back into a solid. The shear shinning is a result of the physical nature of the hydrogel; there are no covalent bonds, only physical interactions holding each individual hairpin together and the supramolecular gel network. So, the hydrogel can break into domains and flow without damaging the network.(Figure 4) When the hydrogel reaches its destination after flow, the domains immediately repercolate back together and form a stiff solid. This has implications for the delivery of various payloads encapsulated inside the hydrogel (28–30). Also, the amphiphilic nature of the hydrogel means that there are domains for both hydrophobic and hydrophilic molecules to be stored.
Figure 2. Cartoon schematic of peptides folding into the β-hairpin conformation under physiological conditions followed by self assembly into a fibrillar network. Reprinted in part with permission from Anal. Biochem. 2017, 535. Copyright 2017 ELSEVIER (25).
Modifications, Implications, and Applications The design history of the MAX peptides is one of modification, both small and large, to improve the hydrogel for a particular application, to further investigate a specific material property, and to study the self-assembly mechanism. This chapter will categorize these various alterations to the peptide sequence, give some context to the goals of the alteration, and show how the alteration affected the structure of the peptide, affected the self-assembly, and affected the ultimate hydrogel material properties. The two major applications in mind for the hydrogel are as a method of payload delivery; such as cells, small molecules, or biological therapeutics; and as a synthetic extracellular matrix to create advanced cell culture models. There are also potential uses of the materials for which both of the above applications are important hydrogel attributes. A recent application being investigated that does 143 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
just that is high throughput drug screening (25). High throughput drug screening is the process of designing disease models that can be tested many thousands of times to quickly screen very large compound libraries. Drug screening tends to rely on liquid handling due to the precision needed when working with such small volumes, and the inclusion of a solid scaffold can greatly complicate matters, However, a shear-thinning and rehealing hydrogel such as those made with betahairpins can be used for both cell encapsulation and culture with defined cell distribution as well as injected with HTS equipment.
Figure 3. Cartoon schematic of the specific packing of β-hairpins within the MAX nanofibril. Reprinted in part with permission from Proc. Natl. Acad. Sci. 2015, 112 (32), 9816–9821. Copyright 2015 National Academy of Sciences (24).
Assembly Early ideas for molecule design based on amino acids include an early study on diblock copolypeptide amphiphiles which tested the gelation ability of lysinexleuciney and lysinexvaliney, an alpha-helix and beta-strand former respectively (31). The resulting materials were able to gel at ~1 weight percent and displayed a rapid recovery of the gel stiffness following a breakdown of the structure by large amplitude oscillations in the rheometer. Although the conclusion was that alpha-helical gelators were slightly better than beta-strands in block copolypeptides, the need for new assembling peptides was clear, leading the way to create the much smaller beta-hairpin MAX peptide. Soon after, a 144 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
paper detailing the MAX1 peptide was published detailing the control of pH over self-assembly and proposed a mechanism for gelation where in a basic solution (pH 9) some of the lysines are neutral allowing the peptide to fold (21). The next study focused on the importance of temperature to the hydrogel system (22). MAX2 and MAX3 were synthesized substituting one (position 7) or two (position 7 and 13) threonines for valine respectively, making MAX2 more hydrophilic than MAX1 and MAX3 more hydrophilic than both. Holding pH at 9 MAX3 was shown to self-assemble at 75 C but unassembled when brought to 5 C, the behavior was shown to be repeatable.
Figure 4. The evolution of the hydrogel network before during and after shear. Reprinted in part with permission from Soft Matter 2010, 6 (20), 5143–5156. Copyright 2010 Royal Society of Chemistry (27).
The effect of salt on self-assembly and the idea of biological applications was a clear next step in the beta-hairpin study. At a solution pH of 7.4 (biological), MAX1 is not able to assemble without the addition of salt. At pH of 7.4 MAX1 has a positive charge from the lysines. Therefore, significant, additional counterions are required to screen those charges before self-assemble can occur. At 400 mM NaCl the peptide rapidly self-assembled to 2000 Pa, at 150 mM NaCl (biological) the peptide assembled to 200 Pa, and at 20 mM NaCl the peptide slowly assembled to 100 Pa. This study showed how salt can affect assembly 145 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
and introduces the idea of how the speed of assembly kinetics can affect the final hydrogel network structure. The faster the assembly kinetics, the more branch points are formed due to defective incomplete collapse of the hairpin hydrophobic faces, thus forming a more stiff hydrogel network even with the same concentration of peptide molecules (32). MAX4 and MAX5 were designed to test the importance of lateral hydrophobic interaction between neighboring molecules valines (33). In MAX1 the valines make both facial and lateral interactions, while MAX4 makes only facial interactions. TEM shows that MAX1 makes very consistent fibrils while MAX4 has many higher order assemblies. A deeper look into the hydrophobic face of the peptide and the importance of branching due to defective hydrophobic collapse focused on the 8 flanking valines of the beta-hairpin replaced by four napthylalanines side chains and four alanines to make LNK1 ((Nal)K(Nal)KAKAK-VDPPT-KAKAK(Nal)K(Nal)-NH2)) (34). The goal was to force the hairpins to stack across the fibrils in a specific manner matching the four bulky napthylalanine side chains from above with the four small alanine side chains from below, forming a lock and key-like steric packing in the hydrophobic core of the fibrils. The design was a success, and fibril branching was greatly reduced. The lack of branching also manifested in the inability of the hydrogels to recover following shear thus revealing that the branch points formed during self-assembly of the MAX family of peptides are critical to the inherent shear thinning and immediate rehealing properties. This work was further expanded with a study that replaced all 8 valines with a variety of other hydrophobic amino acids (35) including valine, aminobutyric acid, norvaline, norleucine, phenylalanine, and isoleucine as l replacements. The most important variable to assembly was found to be hydrophobic content with the more hydrophobic amino acids assembling at lower pH and temperature. The stiffness of the resulting hydrogel was harder to attribute specifically to the molecule structure, as it did not depend clearly with hydrophobicity or beta-sheet propensity. This is because the molecules were found to not simply assemble into a nanofibril with a bilayer of peptide and a hydrophobic core but to assemble in a hierarchical fashion into larger ribbons and fibers. An iterative study looked into the relationship between of the total charge of the molecule and the pH of solution (36). This was done by designing MAX1 modified peptides replacing a single lysine with a glutamic acid at each position, and designing MAX1 modified peptides with altered net charge. This was done to find a peptide that would assemble quickly at pH 7, an important pH for its biological relevance. MAX1 (K15E) was the resultant molecule later named MAX8 and will be discussed at length below. An in-depth study on the evolution of the nanostructure during self-assembly showed two distinct time scales. The first includes fibril cluster formation and intercluster overlap, while the second involves the percolation of fibrillar clusters (32). This has implications for when to include payloads that will be encapsulated in the gel; too slowly during the hydrogelation process and the payload may not be homogenously distributed. Further investigation into the kinetics of assembly comparing the faster assembling MAX8 to MAX1 using neutron scattering to probe the nanoscale networks showed that changing one amino acid to decrease total charge results in a higher storage modulus (stiffness) from increased fibrillar branching 146 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
and physical crosslinks, while the dimensions of individual fibrils did not change (37). The most exact information about the final fibrillar structure is from an NMR study that shows MAX1 assembles in a highly specific way resulting in monomorphic fibrils within a kinetically trapped hydrogel network (24). The resulting fibril can be defined as Syn/Anti with all of the hairpins in the same beta-sheet have their turns on the same edge, and the hairpin in the opposite beta sheet all have their turns on the opposite edge. This arrangement allows the turn sequences in the same beta-sheet to pack decreasing the solvent exposed area, and the terminal valines are able to interact with the turn sequence prolines of their vertical neighbor.
Figure 5. Neutron scattering of vincristine-laden hydrogel with the proposed drug-gel configurations. Reprinted in part with permission from Biomater. Sci. 2016,4, 839-848. Copyright 2016 Royal Society of Chemistry (30).
Antibacterial Properties While the MAX hydrogel was designed with biological applications in mind, it was discovered in the Schneider laboratory to have the fortuitous material property of inherent broad spectrum antibacterial activity. Following this discovery, the bulk hydrogel was challenged with a number of Gram positive, ex Staph, and Gram negative, ex E Coli, bacteria (38). The bacteria were killed so long as the amount of bacteria added did not pass above a certain threshold. The suggested mechanism of destruction is a mechanical tearing of the bacterial inner and outer membranes when the negatively charged bacteria interact with the positively charged hydrogel. This innate mechanism is effective until cellular debris from dead bacteria builds up to create a buffer, preventing new living bacteria from reaching the hydrogel, and overwhelming the antibacterial 147 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
properties. Cocultures of NIH 3T3 fibroblasts and bacteria did not show a negative effect on the fibroblasts proliferation while the bacteria were killed. To further explore antibacterial hydrogels, MARG1 was synthesized replacing lysine with arginine at position 6 and 17 to mimic other antimicrobial peptides and add the guanidinium functional group (39). MAX1 and MARG1 were challenged with MRSA. Both were able to kill the bacteria, but MARG1 was effective up to higher concentrations of the bacteria. Importantly, the bacteria still needed to make contact with the hydrogel for it to be effective. Mammalian mesenchymal stem cell proliferation was not inhibited by culture on MARG1. Cell Scaffolding, Delivery, and Culture With the basics of self-assembly understood, the focus turns more toward applications. The first major biological investigation looked at the cytocompatibility between MAX1 and NIH 3T3 fibroblast cells (40). First DMEM was shown to induce peptide self-assembly and not cause material degradation over time. Next the 3T3 cells were shown to readily attach to the hydrogel when added on top of the preformed gel. The cells were shown to successfully proliferate over 72 hours, even outperforming the control cells grown on polystyrene. Finally, the cells were shown to have almost no effect on the hydrogel stiffness following the 72 hours of growth. MAX8 was designed to improve the biological applicability of beta-hairpin peptides by increasing the speed of gelation at pH 7.4 (41). This was done by exchanging a lysine for a glutamic acid at position 15 lowering the peptides charge state by 2 and allowing the remaining lysine charges to be screened more efficiently (42). This change allowed the hydrogel to form fast enough to homogenously suspend encapsulated mesenchymal stem cells and provided evidence that the cells would remain suspended during injection through a syringe. Having established the MAX hydrogels as cytocompatible in vitro, the next goal was to look at the possibilities of in vivo. J774 mouse macrophages were cultured on MAX1 and MAX8 and their inflammatory response was measured (43). The macrophages were able to successfully proliferate, but displayed no inflammatory response providing evidence that MAX could be successfully implanted in vivo. Small angle neutron scattering and rheology was used to examine MAX1 and MAX8 restoration kinetics following a shear disruption of the network to prepare for delivering cell encapsulated in the hydrogel via syringe (27). The speed of the rehealing process was dependent on the rate and duration of the shear force as well as the stiffness of the hydrogel before shear, but there was no measurable change to the hydrogel nanostructure for any of the shear rates tested, suggesting that the hydrogel breaks into domains that can repercolate together following a secession of shear force. MG63 osteosarcoma cells were encapsulated in MAX8 and underwent a test injection to measure the effect of the injection on cell viability, 3 hours after the injection greater than 95% of the cells were still viable. Polystyrene spheres modified to fluoresce where encapsulated in MAX8 to visualize hydrogel flow through a capillary, mimicking flow in a syringe (26). It was discovered that the gel underwent plug flow; while there was a difference in the velocity of the gel close to the walls of the syringe, decreasing as it the gel 148 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
got close to the wall, the gel in middle area of the syringe flowed at a constant velocity, preventing it from experiencing shear, which has been shown to have a phenotypic effect on cells (44–46). The total cross-sectional area of the capillary that was at a constant velocity depended on the total flow-rate; with an increased flow-rate, the cross-section at constant velocity decreased. This information can be used to protect cells from shear during injection which has been shown to have phenotypic effects on cells. MAX8 was used as a cell scaffold as a part of a high throughput drug screen (25). The hydrogel was modified to include the cell binding motifs, RGDS IKVAV and YIGSR, to simulate extracellular matrix proteins. These modifications to the cellular microenvironment resulted in phenotypic change in the ONS76 medulloblastoma cells making them more invasive and improving stemness. These changes also resulted in the cells responding differently to the drugs when compared to 2D. Another modification that can be made to control the cellular microenvironment is the inclusion of sights susceptible to proteolysis by matrix metalloproteases (47). A series of susceptible hydrogels where designed and synthesized called DP1-4, they were shown to degrade specifically to MMP-13 dependent upon the susceptibility of the individual sequence and the stiffness of the bulk hydrogel. The inclusion of the MMP cleavage sequence was shown to improve cell motility through the hydrogel. Therapeutic Delivery The MAX hydrogels provide a number of useful material properties as a possible delivery vehicle. The ability to undergo triggered self-assembly prior to injection, flow like a liquid without experiencing a breakdown of the local hydrogel network, and immediate rehealing into a solid after a secession of the shear force are all useful properties. When combined with the biological compatibility as well as the tunable nature of the synthetic hydrogel, the material becomes an attractive delivery vehicle. A first delivery study in the Schneider lab looked at three dextran probes of increasing size and their ability to diffuse out of the MAX1 and MAX8 hydrogel networks (28). As expected the relationship between the probe size and the network mesh size had the greatest effect on diffusion rates, providing a way to control the rate via peptide design. A deeper investigation looked into the ability of MAX8 to perform as a delivery vehicle for a more varied selection proteins and how the nature of the encapsulated protein effected the gel network and the proteins diffusion (48). The first thing to note was that the hydrogelation of MAX8 was not impeded by the presence of the protein payloads nor did it have a measurable effect on the hydrogel bulk properties. The release of the positively charged and neutral proteins was largely governed by the size of the protein and its steric integration with the mesh size of the hydrogel network. The release of the negatively charged proteins was impeded by their electrostatic interaction with the positively charged peptide network. Diffusion was tested again on loaded gels following a syringe injection. There was a small effect lowering diffusion rates overall but releasing more of the loaded protein. Taking this information in a therapeutic direction, the neurotrophic growth factors NGF and BDNF were encapsulated in MAX8 to study the possibility of sustained 149 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
local delivery to spinal cord injuries (49). Release rates for both growth factors was shown to be dependent on the weight percent of the hydrogel and the amount of growth factor encapsulated. The rate was also measured to be consistent over 28 days. The loaded gel was used to deliver NGF to PC12 adrenal medulla cells, which responded by growing neurite like extensions. The cells continued to show this phenotypic response to the encapsulated NGF past 28 days of culture, considerably longer than the cells response to NGF in solution which lasted for 3 days. A targeted study was done encapsulating curcumin as a potential chemotherapeutic (29). Curcumin is a challenging therapeutic molecule because of its hydrophobicity and short half-life. The encapsulation of varying concentrations curcumin into the MAX8 hydrogel was shown to have minimal effects on the hydrogel bulk properties and released from the gel at a consistent rate over two weeks while and inducing cell death in DAOY medulloblastoma cells. A more conventional chemo therapeutic, vincristine, was also encapsulated in a later study to study release and effectiveness (30). The loaded vincristine was shown to release over 28 days and still be able to induce cell death in DAOY cells at day 28, considerably longer than its half-life of 4 days in aqueous solution would suggest possible.(Figure 5) This is another example of the protective benefit the amphiphilic hydrogel has for hydrophobic compounds. More recent delivery studies include the use of MAX1, MAX8, and HLT2, a peptide modified to have a lower net positive charge, to deliver DNA and stimulate an immune response in mice (50). It can be a challenge to deliver whole, undegraded DNA in a therapeutic application. It was hypothesized that a beta hairpin hydrogel would be able to protect its DNA cargo en route to its destination (51). All three gels were able to retain greater than 90% of the encapsulated DNA over two weeks. After injection into mice MAX8 and HLT2 gels each showed an increase in lymphocyte proliferation, but only HLT2 had large amounts of infiltrating cells, resorption of the hydrogel, and growth of new tissue. These results suggest that DNA was unable to be released from the highly charged MAX1. Major Modifications While the beta-hairpin conformation is robust to arm modification, the turn sequence and the hydrophobic face of the folded peptides are more fundamental to the existence of the hairpin structure. A study into the turn sequence focusing on the replacing the d-proline with an l-proline resulted in no hydrogel structure but instead the formation of twisted beta-sheet-rich ribbons with high stability to solution changes after assembly, a large change due to changing the chirality of one amino acid (52). MAX6 was designed to test the how the inclusion of a charged amino acid on the hydrophobic face would affect folding by exchanging a valine for a glutamic acid at position 16. MAX7 tested the effect of exchanging a valine with a cystine on folding, which had little effect. Both of these molecules were used to explore the addition of more molecular controls to triggering selfassembly (53). In particular, the cystine was used to create MAX7CNB through the addition of a photocage that has a negative charge under basic conditions. The molecule would not self-assemble until the photocage was removed by UV light. 150 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
The resulting hydrogel was also shown to support NIH 3T3 fibroblast proliferation. A similar peptide called MLD was designed by replacing a lysine at position 5 and 18 with a lysyl sorbamide and adding an extra lysine at the beginning and end of the peptide for a total of 22 amino acids. The sorbamide groups can be covalently crosslinked using UV following self-assembly of the hydrogel network. After the network physically self assembles, the UV induced polymerization increased the stiffness of the gel 2.5 fold, giving another tool to tune the hydrogel. Strand swapping peptides were designed by synthesizing hairpins with uneven sets of valine lysine pairs flanking the turn sequence (54). SSP1 (VK)2-VDPPT-(KV)6- NH2 had two sets of valine lysine pairs on one side and six pairs on the other, this meant that the resulting hairpin was two pairs long with a dangling four pair domain that could beta-strand swap with another peptide forming a facially amphiphilic dimer. SSP2 (VK)3-VDPPT-(KV)5NH2 was similarly designed with three pairs in the hairpin and two pairs dangling. The normal self-assembly pathway resulted in fibrils. However, the SSP1 fibrils twisted while SSP2 fibrils did not. A further study created SSP3 (VK)5-VDPPT-(KV)3- NH2, a peptide identical to SSP2 with the strand asymmetry switched to have the longer stand on the N-terminus (55). All three peptides made individual fibrils with a four peptide cross-section instead of the normal two peptide cross-section in the MAX peptides. This is because the extra beta strand length in a folded peptide requires a partner peptide on the same face of the nanofibril to satisfy the possible hydrogen bonds available in the extra beta-strand. After folding and hydrophobic collapse, the individual fibrils contain a four peptide cross-section. The fibrils either twisted, remained straight, or laminated, and each network had different stiffnesses. A three stranded peptide with two turn sequences called TSS1 was designed to make two hairpins during the folding process (56). It had similar material properties of triggered self-assembly, shear thinning, and rehealing, but the hydrogel had a greater stiffness than unmodified MAX1 giving a way to control stiffness not connected to weight percent or speed of assembly. A second method of stiffness control unrelated to kinetics or weight percent was found while studying the enantiomer of MAX1 called DMAX1 synthesized using d amino acids (57). It was originally of interest as a way to prevent enzymatic degradation, and when gelated alone, behaved nearly identically to MAX1 in terms of material property. But, when the two peptides were mixed together the resulting hydrogel was much stiffer. The greatest increase, a four fold increase, occurs when the two peptides were mixed one to one. If the ratio increased in favor of one peptide or the other the increase in stiffness was lessened. A final point on use of L or D amino acids, the DP found in the hairpin turn sequence, while a critical portion of the molecule design, makes bacterial expression of the peptides difficult. Three peptides were designed that would be expressible by bacteria with redesigned turn sequences called EX1 ((VK)4-VPDGT-(KV)4-C02H), EX2 ((VK)4-VPIGT-(KV)4-C02H), and EX3 ((VK)4-YNGT-(KV)4-C02H) (58). Each peptide was shown to be suitable for bacterial expression with all three peptides able to self-assemble, shear-thin and reheal with EX3 being the most stiff hydrogel. TEM of the fibrils showed that EX1 was more heterogenous than the other two, possibly from the negatively charged aspartic acid added to the hydrophobic face. MBHP 151 Horkay et al.; Gels and Other Soft Amorphous Solids ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
(VKVKVKV-CGPKEC-VKVKVKV-NH2) is also a peptide designed around a modified turn sequence. It relies on the binding of a heavy metal ion to form the individual beta-hairpins (59). The addition of the two cystines flanking the turn sequence provide a location for the metal ion to bind and force the peptide to fold. The nanostructure of the network depended on the type of metal bound, twisting or laminating fibrils with some making very stiff final networks.
Conclusion The development of the beta-hairpin peptide was one of careful choices and simultaneous serendipitous discovery. The hydrogels resulting material properties make it a powerful tool for both therapeutic delivery and complex cell culture. The growing list of peptide modifications and design tools means it can be tailored to fit many specific situations currently and in the future.
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