Rational design of fiber forming supramolecular structures

Abstract

Recent strides in the development of multifunctional synthetic biomimetic materials through the self-assembly of multi-domain peptides and proteins over the past decade have been realized. Such engineered systems have wide-ranging application in bioengineering and medicine. This review focuses on fundamental fiber forming α-helical coiled-coil peptides, peptide amphiphiles, and amyloid-based self-assembling peptides; followed by higher order collagen- and elastin-mimetic peptides with an emphasis on chemical / biological characterization and biomimicry.

Introduction

Numerous advances in biomimicry have transformed our understanding of the fundamental requirements for recapitulating the extracellular environment. Mimicry of supramolecular structures that provide an instructive scaffolding for cells, tissues, and organs is a mainstay for the fields of biomaterials, materials chemistry, and tissue engineering.^1–5 Through advances in nanotechnology and peptide chemistry it is now possible to recreate the fundamental building blocks of mammalian life—such as collagen and elastin in their native hierarchical structures.^6–8

Peptide-based materials stand at the forefront of several tissue engineering strategies.^9–18 Owing to the modular nature peptide-based materials, a variety of different moieties can be introduced.¹⁹ These moieties can guide self-assembly, bioactivity or both—as discussed in this review. Peptide-based materials originally focused on pharmaceutics. With a better understanding of secondary protein structure and how to tailor supramolecular self-assembly, short polypeptides have been used for a variety of applications from drug/ growth factor mimicry,²⁰ drug delivery,²¹ inflammation modulation,²² and orthogonal self-assembly with loaded liposomes.²³ Additionally, as detailed in this review, stable large scale hydrogels formed by non-covalent interactions of these peptide-based materials allow recapitulation of a nanofibrous extracellular matrix (ECM) mimetic scaffold.^19,24 Variants of these scaffolds have allowed for excellent cellular spreading in vitro and rapid infiltration without the formation of fibrous capsules in vivo.^20–23 Together, these biological and mechanical cues inform the design and application of a host of peptide-based materials, whose organization and core structure are analyzed in this review.

Two specific approaches can be used to fabricate novel biomaterials: top-down and bottom-up approaches.²⁴ The former utilizes understanding global structure with subsequent deconstruction and mimicry of macromolecular constituents. The top-down approach oftentimes fails at achieving specific structure and assembly. The consensus in the field is that construction of larger subunits of proteins and organized tissue requires a building-block bottom-up approach where assembly is dictated at the molecular level. De novo engineering with inspiration from nature allows programmed folding and self-assembly. Ultimately, secondary and tertiary protein structure dictate molecular arrangement of bioinspired materials.¹⁷ Thermodynamic, entropic, and stereochemical factors guide non-covalent self-assembly of supramolecular structures that dictate mechanical and biological functionality.^3,25–27 Ranging from alternating hydrophobic and hydrophilic amino acids that create facial ampiphiles^28–30 to Xxx-Yyy-Gly sequences that predispose α-helix formation, nature has evolved a set of rules that govern arrangement at the molecular, nano, micro, meso, and macroscales.^31–33 The rational design of fiber forming biologically inspired materials, requires an understanding of these interactions for directed molecular self-assembly.³⁴

α-Helical coiled-coils

α-Helical building blocks are fundamental supramolecular structures which comprise the majority of biological tissue.⁴ By rationally designing sequence-to-structure relationships between α-helices, rules for engineered protein folding and assembly can be achieved. At the amino acid level, Woolfson's group reports the predisposition of specific amino acids alanine, glutamine, glutamic acid, and lysine for a-helices over threonine and valine for β-structure.^17,35 Furthermore, given the small size and ability to pack efficiently, glycine and valine are commonly found at α-helical turns.⁴ The canonical α-helical coiled-coil architecture involves a repeating abcdefg heptad that assembles to form an amphipathic α-helix secondary structure, and further into a left-handed dimer.³⁵ Noncanonical α-helical coiled-coils do not contain this characteristic heptad repeat, and as a result, do not necessarily form dimeric supercoils.³⁵ Similar to the majority of self-assembling peptide structures, non-covalent hydrophobic interactions between the side chains of different α-helices drive folding. Consequently, hydrophobic amino acids are usually spaced three to four amino acids apart to satisfy the hypothesized 3.6 amino acid α-helix geometry.^17,36 Most commonly, coiled-coil sequences take the form (HPPHPPP)_n≥4, where hydrophobic (H) amino acids are at the a and d positions and polar (P) amino acids at all other positions.³⁷

Pioneering this structure of self-assembling peptides, in 1998 Petka's group produced an early synthetic coiled-coiled α-helix that reversibly self-assembled into a hydrogel.³⁸ This peptide used a triblock architecture with two flanking leucine zipper motifs separated by a flexible alanylglycine-rich repeat. Each terminal leucine zipper comprised the characteristic α-helical abcdefg heptad repeat, with Leu frequently at positions a and d. Hydrophobic interactions between each of the amphipathic helices promoted the assembly of coiled-coil dimers between peptide strands, promoting aggregation, and subsequent gelation. Similarly, elevating pH or temperature disrupted coiled-coil aggregation, and thereby melted the hydrogel to a viscous liquid state.³⁸ Building upon this work, various α-helical coiled-coil systems have since been developed.^37,39–43 In 2000 Woolfson's group designed the first “sticky-ended” heterodimer that promoted the formation of long fibers.³⁷ Since then they have demonstrated the design of the MagicWand peptide, a single peptide that assembles to nanoscale fibers. This architecture directed the staggered assembly of α-helices to promote coiled-coil fibrillogenesis by incorporating an anionic core region with cationic flanking regions.⁴⁴ Another system they have developed promotes longitudinal fiber assembly of α-helical coiled-coils by substituting the b and c positions of the heptad repeat with oppositely charged residues to form complementary offset-register dimers with “sticky-ended” overhangs.⁴⁵ Ionic interactions between helices promoted strong fibril aggregation resulting in precipitation, limiting the utility of this system.

More recently, work on engineered α-helical architectures has shifted towards fiber-forming biocompatible hydrogels for application in mimicking the extracellular milieu.^42,46 For example, these hydrogel scaffolds are promising for the controlled delivery of drugs as well as supports for cell growth and tissue engineering.^17,47 Designer heptad repeats with altered b, c, and f positions substituted with either Ala or Glu residues were used to form weaker and more general hydrophobic interactions and hydrogen bonds between fibrils.⁴⁶ Instead of thick fiber aggregation and precipitation, a hydrogel with thinner, more flexible fiber bundles assembled. The incorporation of hydrophobic interactions was useful in controlling gelation, as an increase in temperature resulted in a stronger gel when the altered b, c, and f positions were Ala substituted. Cytocompatibility of hydrogels was tested by seeding rodent adrenal phechromocytoma cells and measuring neutrite outgrowth.⁴⁸ Hartgerink's group has demonstrated the design of blunt-ended coiled-coiled architectures that self-assemble into long nanofibers and form a hydrogel.⁴² They have shown that the thickness of fibers can be controlled by varying the amino acids found at the b, c, and f positions of the heptad. Moreover, charged Lys residues at the periphery increased repulsion between coils resulting in thinner fibrils, while non-covalent interactions between hydrophilic amino acids promoted thicker fiber bundles.⁴²

β-Hairpin peptides

β-Secondary structures constitute another underlying motif for self-assembly that incorporates both distinct material properties, biofunctionality and are commonly known for their preponderance in pathological amyloidosis.⁴⁹ These peptides usually integrate alternating hydrophobic (H) and polar (P) residues (HPHPHP)_n and assemble to form facial amphiphiles with opposing hydrophobic and hydrophilic faces. As a result of non-covalent intermolecular van der Waals forces and hydrophobic packing, these secondary structures further assemble into higher order structures, including fibers, micelles, bilayers, and extended β-sheets. One important subset of β-based peptides is the β-hairpin motif. These sequences include two antiparallel β-strands that are joined by a tetrapeptide type-II β-turn.^48–50 The β-turn is one of the most common peptide secondary structures and is largely used for directional change in a polypeptide sequence.⁵¹ In the β-hairpin architecture, this loop, along with the flanking β-strands, are critical components for the conformation and stability of the overall structure.⁵² Alternating hydrophobic and hydrophilic amino acids populate the remaining peptide flanks, forming amphiphiles, which can then associate with other amphiphiles both facially and laterally to promote a nanoscale fibrillar structure and hydrogel network. In these strands, amino acids are organized to maximize interstrand hydrogen bonding and side-chain interactions.⁵¹ In 1993 Blanco et al. reported Nuclear Overhouser Effect (NOE) NMR showing that the sequence YQNPDGSQA had a significant population of isolated β-hairpins in aqueous solution attributed to the high turn probability of the central residues.⁵² Schneider et al. in 2002 described the self-assembly of a 20-residue peptide composed of a (-V^DPPT-) β-turn with alternating Val and Lys residue flanking regions, MAX1.⁵³ MAX1 exhibited pH triggered self-assembly into a hydrogel under basic conditions. Val residues were used to promote hydrophobic collapse into a β-sheet architecture while Lys residues were incorporated to control gelation—at sufficiently low pH, electrostatic repulsion between positively charged Lys residues resulted in β-hairpin dissolution and subsequent hydrogel dissolution.⁵³ Pochan et al. have since designed variations of MAX1 and have demonstrated precise control over hydrogelation of these peptides by changing conditions such as temperature,⁵⁴ pH,⁵⁵ ionic strength,⁵⁶ and light.⁵⁷ They have shown a rise in temperature triggered gelation by inducing a stronger degree of hydrophobic collapse between Val residues,⁵⁴ while an increase in ionic strength screened electrostatic repulsion between Lys residues.⁵⁶ As a result of built-in physical cross-linking, these gels also have a remarkable ability to shear-thin.⁵⁸ Demonstrating the tunability of the system for physiologic condition, more recently, Haines-Butterick et al. have designed MAX8, substituting a Lys residue in MAX1 for Glu, which decreases the net positive charge, allowing MAX8 to gel at physiological pH much more quickly.⁵⁹

β-Sheet peptides

The principles underlying β-hairpin self-assembly are fundamentally similar to the structural organization motifs of self-assembling peptide amphiphiles. These designs generally involve the facial self-assembly of amphiphilic peptides to form long, high-aspect-ratio nanofibers.⁶⁰ Most commonly, these synthetic peptides comprise a region of β-sheet forming amino acids coupled with a solubilizing region that allows fiber and hydrogel formation in aqueous solution.^61,62 These assemblies provide a facile method for bioactive sequence incorporation to promote the adhesion / infiltration of cells.^62–64 There exist several examples of β-sheet-based peptide amphiphilies including Collier and Messersmith's Q11 peptide with alternating polar and aromatic residues,⁶⁵ and Goeden-Wood et al.'s (AEAEAKAKAEAEAKAK)₉ peptide which formed strong nanoscale fibrous hydrogels.⁶⁶ Jun et al. has demonstrated fiber-forming and hydrogelating systems with built-in bioactive functionality through the development of “ionically complementary” peptides, known as Lego peptides.⁶⁷ The formation of complementary ionic pairs between amino acids from different peptide chains can lead to self-assembling, electrostatically stable, higher-order aggregates, such as nanofibers or globular structures.²⁴ Zhang et al.'s first peptide of this type, EAK16-II was composed of complementary (AEAEAKAK)₂ sequences and associated into a β-sheet membrane structure through hydrophobic interactions and the formation of ionic bonds between oppositely charged Glu and Lys residues.^68,69 This stable self-complementary 12-amino-acid peptide gelled upon the addition of salt into a membranous structure, and demonstrated stability in heat, reducing conditions, and pH variations.⁶⁹ Derivations of this peptide—EAK16-I (nanofibers) and EAK16-IV (globular structures)—comprising identical amino acid compositions but with different sequence organizations were also demonstrated.⁶⁷ Of particular note—RADA16—has arguably gained much repute and is currently marketed under the name Puramatrix® by BD Biosciences.⁷⁰ This peptide substitutes Glu and Lys residues for Arg and Asp and undergoes molecular self-assembly into nanofibers to form a hydrogel scaffold.⁷¹ The reader is directed to the following articles for more information on self-assembly, cytocompatibility and in vivo studies of Zhang and co-worker's work.^3,8,70,72,73 Aggeli et al. have reported the development of self-assembling amphiphilic β-sheet tapes, capable of forming hydrogels composed of nanometer-long fibers.^74–76 Fundamentally, these peptides aggregate to form antiparallel β-sheet tapes through cross-strand weak attractive forces.⁷⁴ To prevent the precipitation of aggregated β-sheet structures, lateral, one-dimensional assembly is ensured by incorporating molecular recognition mechanisms into side chain interactions: for example, aromatic residues can be used to provide intermolecular recognition by π–π stacking interactions. Moreover, strong solvent-to-surface interactions are ensured to control solubility in solvents of different polarity. The earliest of the group's designed peptides include the 24-residue peptide K24 and 11-residue peptide DN1, which self assemble in non-aqueous and aqueous solvents, respectively.⁷⁴ K24 was designed with an amphipathic primary sequence, with polar residues at the termini and nonpolar and aromatic residues in the core region, which was hypothesized to control one-dimensional β-sheet propagation. Similarly, the much shorter DN1 assembled through intermolecular π–π, hydrophobic, and ionic interactions. In both cases, association between aromatic amino acid residues significantly contributes to the peptides' self-assembly properties. More recent work by their group has demonstrated the self-assembly of stable β-sheet tapes without the use aromatic π–π interactions.⁷⁵ 7mer and 9mer peptides P₇-6 and P₉-6 contained predominantly aliphatic Leu residues on their hydrophobic faces, and still underwent a transition from monomeric random coils to β-sheet tapes at higher peptide concentration and neutral pH. At lower pH, protonated side chains decreased intermolecular attraction, and thus increased the critical concentration necessary for self-assembly. This study also demonstrated the increased propensity for self-assembly in polar organic solvents such as methanol, and the lower critical concentration needed for the self-assembly of longer peptides.

Recent advances in β-sheet forming systems have incorporated ancillary domains for greater control over fiber formation and hydrogelation. An important example of this has been demonstrated by Hartgerink's group with multi-domain ABA triblock peptides that self-assemble to form highly ordered nanofibers in a hydrogel scaffold.^{61,62,64,77–79} The self-complementary alternating hydrophobic–hydrophilic peptide cores pack to exclude hydrophobic residues from the aqueous environment, and additional peptides can then assemble laterally by intermolecular backbone hydrogen bonding, forming high aspect ratio nanofibers. Because such assembly can continue indefinitely until all monomeric peptides are depleted, flanking positively charged A blocks at neutral pH are incorporated to produce electrostatic repulsion between dimers. These flanks comprise 0-4 frustrated, positively charged lysine residues that counter the B block's strong affinity to associate, thus preventing complete aggregation and subsequent precipitation. Hydrogelation is optimized by the addition of multivalent ions that screen charge repulsion allowing a remarkable level of control over fiber formation and hydrogelation.⁷⁷ Demonstrably the sequence K_n(QL)_mK_n showed that varying the n/m ratio favored either self-assembly or disassembly; and only when the forces promoting self-assembly and opposing charge repulsion were balanced did the formation of controlled length nanofibers become possible.⁷⁷ Further, various derivatives of the K_n(QL)_mK_n sequence, producing similar fiber-forming self-assembling hydrogels with added functionality have been developed. For example, a sequence in which Gln residues were substituted with Ser residues, K₂(SL)₆K₂, demonstrated a significantly greater storage modulus and greater degree of shear recovery than its Gln counterpart.⁷⁸ Such a system with highly tailorable viscoelastic and shear thinning properties has great implications with respect to injectable hydrogels for drug delivery and regenerative medicine.^{20–23,61,64,78} In addition to conventional alternating hydrophilic–hydrophobic core regions, Hartgerink's group has demonstrated the self-assembly of triblock multi-domain peptide with an alternating hydrophilic-aromatic core region.⁶¹ Recently they described a novel aromatic self-assembling peptide amphiphile. In contrast to the above-mentioned mechanism for self-assembly, the introduction of aromatic amino acids changes the packing characteristics between peptide chains and allows for the possibility of interstrand π–π stacking interactions. Interchanging Leu residues for aromatic Phe, Trp, and Tyr residues while keeping hydrophilic residues unchanged caused a significant change in fiber morphology when observed with electron microscopy. However, despite the large steric strain afforded by bulky aromatic residues, in all cases, these multi-domain peptides self-assembled and retained a basic nanofibrous structure.⁶¹

Amyloid-based self-assembly

Another class of β-based peptide biomaterials can be engineered from naturally occurring self-assembling amyloid fibers. In natural systems, amyloid fibers that arise as inappropriately folded polypeptides ultimately manifest as insoluble protein aggregates that have been linked to a variety of human diseases, including amyloidosis and a variety of neurodegenerative disorders.^80,81 Energetically, this occurs as conformational shifts from monomeric unfolded intermediates into β-sheet-rich structures is preferred. Most commonly, cross-β amyloid structures comprise β-sheet peptides organized into parallel or antiparallel β-strands. Two or more β-strands facially assemble to produce fibrils, which are stabilized by hydrogen bonds along the fibril axis and side chain interactions between fibrils.⁸² These interactions are so prevalent that amyloid formation is not completely sequence specific: amyloid formation can be induced by seeding fibrils with the same or unrelated proteins.⁸³ It has been suggested that with sufficient time, all well-folded proteins undergo an irreversible structural transition to a “correctly-folded” aggregated β-sheet structure, which represents a global minimum in Gibbs energy for protein folding.⁸⁴ In engineered systems, however, these supramolecular assemblies, which range in states from liquid crystals to rigid nanofibers, can lead to synthetic biomaterials that introduce biological function and tailored mechanical properties.⁸⁵ Small oligomers of peptides serve as nucleation points for further aggregation and ultimately fibril formation, dictated by non-covalent bonds, notably hydrogen bonds,⁸⁶ hydrophobic interactions,⁸⁴ and π–π stacking.⁸⁷ One of the early examples of this architecture was designed by Hecht et al., who demonstrated that simple alternating patterns of hydrophobic and polar amino acids resulted in aggregation between amphiphilic β-strands, forming β-amyloid fibers.⁸⁸ More sophisticated amyloid-based fiber-forming and hydrogelating biomaterials have since been designed. Notably, the Nilsson group has utilized π–π and hydrophobic side-chain interactions to demonstrate the formation of self-assembled materials inspired by amyloid materials.^89–91 To set the stage for his future work, Nilsson demonstrated that aromatic residues and π–π stacking interactions were not strictly necessary for amyloid formation with the amphipathic sequence (FKFE)₂.^89,91 Instead, residues with sufficient hydrophobicity or β-sheet propensity could drive amyloid fibril formation. When non-aromatic, highly hydrophobic cyclohexylalanine was substituted for phenylalanine residues, self-assembly still occurred and even exhibited enhanced hydrogelation properties. Using these principles, Nilsson has developed modified designs with wide-ranging applications as biomaterials. For example, a designed peptide sequence with the (FKFE)₂ motif and flanking cysteine residues takes on a cyclic structure when intermolecular disulfide bonding is present, but can undergo self-assembly into a linear β-sheet conformation and hydrogel with the addition of a reductive trigger.⁹² Similarly, the Nilsson lab has also demonstrated the design of the coassembly of enantiomeric L-/D-peptides into rippled β-sheet fibrils with enhanced viscoelastic hydrogel properties.⁹³ Other recent examples of bioinspired self-assembling amyloid peptides includes work done by the Guler group. They have demonstrated the formation of a hydrogelating nanofiber scaffold with amyloid characteristics from oppositely charged peptides -E-FFAA-E- and -K-FFAA-K- at neutral pH.⁹⁴ In addition to the tailorability of this peptide architecture with respect to fiber formation and hydrogelation, engineered amyloid fibrils can also be chemically modified by incorporating functional peptide sequences.⁸⁵ The reader is directed to Bowerman and Nilsson for more details.⁹⁵

Peptide amphiphiles

In addition to facially amphiphilic β-based self-assembling peptides, a class of peptide amphiphiles with appended alkyl chains have demonstrated various applications for tissue regeneration and bioengineering.^28,60,96 Pioneered by the Tirrell group, in 1995, a diblock system incorporating a long-chain dialkyl-ester lipid tail and collagen-model peptide head-group was used to form a biologically active cytocompatible evenly dispersed bilayer.⁹⁷ Since then, the Stupp lab has continued with the development of these straight-chain amphiphilic sequences. These peptide amphiphiles are composed of 4–5 regions: region 1 is composed of a long alkyl chain to promote hydrophobic collapse of individual molecules; region 2 largely drives the lateral association of peptide amphiphile molecules through either a β-sheet forming segment stabilized by intramolecular backbone hydrogen bonding or covalent disulfide bonding; region 3 comprises a flexible glycine spacer or charged amino acids for solubility; and region 4 and 5 are functionalized to contain the specific peptide epitope for biological function or signaling. Hartgerink et al., in 2001, produced a novel self-assembling peptide-amphiphile that formed cylindrical micelles and a nanoscale fibrous scaffold with properties similar to those of the ECM.²⁸ This peptide contained the characteristic peptide amphiphile motifs, with regions 4 and 5 containing a phosphorylated serine residue and arginyl-glycyl-aspartyl (RGD) epitope to promote hydroxyapatite mineralization and cell adhesion, respectively. Ultimately, this peptide amphiphile demonstrated the pH-triggered assembly of a chemically robust nanofiber scaffold that could direct ordered hydroxyapatite mineralization and support cell growth. Further studies on this peptide have shown conditions for triggering self-assembly by pH, ionic strength, and concentration, as well as varying amino acids and the alkyl chain for nanofibers of varying morphology and bioactivity.⁹⁸

Other examples of designed peptide amphiphiles that self-assemble to form nanoscale fibers include the pH-induced assembly of hydrogel C(12)-GAGAGAGY based on silk fibroin,^99,100 aromatic Fmoc utilizing hydrogels,¹⁰¹ and even binary mixtures of peptide amphiphiles containing oppositely charged residues.¹⁰² Advances in this facile design allow tailoring the material properties of peptide amphiphile forming hydrogels.¹⁰³ A change in the position and increase in the number of Val residues were reported to form stronger, stiffer hydrogels, while an increase in Ala residues decreased the mechanical stiffness. Fourier transform infrared spectroscopy suggests that this effect is presumably due to the alteration in the alignment of hydrogen bonds along the long axis of the peptide amphiphile fibers: Val residues were hypothesized to form tightly packed, stiff β-sheet micelles/fibers, while the Ala-residue-populated β-sheet cores could not form hydrogen bonds as effectively, leading to more disordered peptide amphiphile micelles with twisted geometries.¹⁰³ Not only does this work demonstrate the significance of strong, ordered intermolecular hydrogen bonding in the mechanical properties of the resulting hydrogel, but also the potential to create softer hydrogels as potential injectable materials. More recently, work on nanofibrous peptide amphiphiles has focused on incorporating a variety of bioactive epitopes for biomaterials with tailored functionality. Peptide amphiphiles containing the neuron lineage driving IKVAV epitope have been developed,¹⁰⁴ as well as, an injectable vascular endothelial growth factor-mimetic peptide amphiphile with pro-angiogenic properties capable of restoring blood flow in a hind-limb ischemia model.¹⁰⁵ Moreover, peptide amphiphiles containing the heparin binding domain,^106,107 growth factor release domains,^108,109 hydroxyapatite nucleation domains,^110,111 RGD cell adhesion motif,^112–114 and cell-apoptosis-promoting regions for cancer treatment¹¹⁵ have been reported. The reader is directed to reviews by Webber et al., Cui et al., and Matson and Stupp for details.^5,60,116

Collagen-mimetic peptides

Building upon fundamental α- and β-secondary structures, a variety of other secondary structures have been explored as self-assembling biomaterials. The prime example of this has been recent work in the study of developing synthetic collagen mimetic peptides (CMPs).^117,118 These short peptide strands mimic natural collagen's Xxx-Yyy-Gly repeating triplet. Here, short-chain polypeptides, consisting of approximately 24–36 amino acids, self-assemble into triple helices and can further mimic all stages of natural collagen's multi-hierarchical self-assembly.^6,119 Most commonly, proline and 4-hydroxyproline occupy the Xxx and Yyy positions of natural collagen, while recent studies have begun to investigate self-assembling CMPs with a variety of amino acid mutations.³⁴ This repeating triplet plays a role in stabilizing triple helix formation. Glycine, the only necessary amino acid in the collagen-repeating motif, provides a compact methylene side chain that is oriented towards the interior of the triple helix and allows for the tight packing of individual polypeptide strands.¹²⁰ This close proximity facilitates inter-strand hydrogen bonding, in which the amine of glycine acts as a hydrogen donor to the carbonyl of proline on an adjacent polypeptide strand.^1,121 This further stabilizes triple helix assembly. In addition, the side-chain rings of proline and hydroxyproline have low degrees of freedom, contributing to the rigidity of the collagen backbone.^122,123 Inter-strand salt bridges between charged residues can also serve to stabilize peptide strands and triple helix formation.¹²⁴ Individual tropocollagen molecules then pack both linearly and laterally to form collagen nanofibrils.¹²⁵ Assembly can further continue to produce triple helices that pack into nanofibers, and finally compose a hydrogel scaffold.⁹⁶

Self-assembly of the first generation CMPs largely stop after triple helix formation, limiting their widespread use. Initial studies have shown the substantial stabilizing role of electrostatic interactions, laying the foundation for higher-order structures similar to natural collagen. Early work by Brodsky's group studied homotrimeric CMP systems. Venugopal et al. prepared a collagen-like sequence (POG)₄(EKG)(POG)₅ in 1994.¹²⁶ This homotrimeric peptide self-assembled into a triple helix, and showed an increase in thermal stability when all ionizable side chain residues were charged, suggesting the stabilizing role of electrostatic interactions between ion pairs. To corroborate these results, a (POG)₁₀ peptide with uncharged amino acids displayed the least stability at neutral pH, due to charge repulsion between ionized C- and N-termini.¹²⁶ Similarly, at higher pH, in which N-termini were uncharged, the peptide gained about 2℃ of stability in melting experiments.¹²⁷ Further work by Chan et al. has demonstrated the dependence of triple-helix stability on the identity, position, and environment of charged residues.¹²⁸ They embedded a collagen G-Y-X triplet in a host Ac(GPO)₃-GXY-(GPO)₄-GG-NH₂ sequence and substituted the Xxx and Yyy positions with ionizable residues. When the Yyy position was substituted with Glu, Asp, Arg, and Lys residues and the Xxx position with Pro, a significant range of triple helix thermal stability was observed. In contrast, when the same substitutions were done in the opposite manner, only a narrow range of thermal stability was noticed. This was attributed to the Xxx position exhibiting a more favorable side chain orientation, in which they point outward in optimal directions for minimizing charge repulsion. Conversely, ionizable residues in the Yyy position appear to be slightly destabilizing due to potential charge repulsion between residues.¹²⁸ Kotch and Raines, in 2006, further demonstrated the self-assembly of a cysteine-containing synthetic collagen triple helix.¹²⁹ Cysteine disulfide linkages between peptide chains served as a means for linking the individual strands into a natural collagen mimetic triple helix. These covalent linkages between strands offset the strands and controlled their register. These “sticky ends”, where the peptide is offset by a number of amino acids, allow additional peptide strands to add end-to-end, elongating the triple helix and driving fiber formation as previously demonstrated by Woolfson and others.^37,130 These charged, unpaired flanking sequences drive self-assembly by satisfying more and more charge pairs with each additional triple helix added. Examination by atomic force and electron microscopy revealed the self-assembly of one-dimensional nanofibrils of 20–120 nm in length, which may have potential as synthetic biocompatible materials.¹²⁹ Advanced collagen mimetic systems have revealed the self-assembly of CMPs into triple helices without the necessity of covalent linkages. In 2007 Chaikof's group developed the zwitterionic fiber-forming 36 amino acid sequence (PRG)₄(POG)₄(EOG)₄. This peptide self-assembled into a triple helix and subsequently nanofiber, without further assemble into a hydrogel.³³ More recent work on hierarchical constructs that are capable of mimicking both natural collagen's higher order fibrillation as well as the biological effects of specific natural tissue scaffolds, and potentially a variety of other novel functions, can be architecturally designed by incorporating specific short-peptide features into a single multi-domain peptide.⁶ For example, Cejas et al. demonstrated this by adding C-terminal phenylalanine and N-terminal pentafluorophenyl flanks to a (Pro-Hyp-Gly)₁₀ CMP. Their system produced micrometer-length fibers with natural collagen-like properties, noting that without incorporation of aromatic flanking regions, fibrillation could not have been achieved.¹³¹ Gottlieb et al. produced conductive synthetic nanowires tens of microns in length by conjugating the collagen mimetic sequence pentafluoro-F-(GPO)₄-GPK-(GPO)₅-F with gold nanoparticles.¹²⁷

In 2007 the Hartgerink group demonstrated the development of a group of electrostatically stabilized ABC collagen mimetic heterotrimers.¹³² These heterotrimeric systems were unique allowing tailored substitution in one, two, or all three peptide chains. This study demonstrated strategies for designing heterotrimers with separate net positive, neutral, and negative strands that assemble into triple helices with net neutral charge. Further, this study suggested that the formation of an Asp-Lys charge pair within a triple helix provides equivalent stability to helices containing collagen's characteristic Pro-Hyp-Gly triplet, which provides a foundation for designing more sophisticated collagen mimetic systems. They then demonstrated the first ABC heterotrimeric collagen mimetic system that self-assembled utilizing only supramolecular interactions,¹³³ a single-register ABC heterotrimer stabilized by electrostatic interactions,^134,135 and design strategies for designing collagen mimetic homotrimers,² AAB heterotrimers,² single-composition ABC heterotrimers.¹³⁶ This work has allowed them to determine design principles for register- and composition-controlled collagen mimetic homotrimers and heterotrimers. The work on CMPs from triple helix, to nanofiber, to hydrogel led to the design of the peptide (PKG)₄(POG)₄(DOG)₄.⁶ This tri-block peptide (+,n,−) is unique since it is a homotrimeric system comprised of peptides with a net neutral charge that incorporates collagen's proline-hydroxyproline-glycine repeating unit in the central domain. Moreover, assembly is driven by the presence of stabilizing inter-strand side chain interactions between lysine and aspartate residues. These salt-bridge hydrogen bonds between oppositely charged ionized residues serve to stabilize a sticky ended triple helix formation, which nucleates fiber formation by allowing additional peptide strands to add end-to-end elongating the triple helix. As a result, self-assembly of KOD nanofibers is observed, which displays the characteristic triple helical packing of natural collagen fibrils. Current work with this synthetic collagen is focused on cytocompatibility and utility as a hemostat.^6,137

Elastin-mimetic peptides

Similar to CMPs that exhibit tissue-like mechanical properties and architectures, elastin-mimetic peptides have been developed to mimic native tissue resilience for example in blood vessels, the lungs, and skin.^138,139 Natural elastin is found as a cross-linked protein comprising alternating hydrophilic polar domains rich in Ala and charged Lys necessary for chemical cross-linking, while the hydrophobic nonpolar domain, rich in Pro, Gly, and Val, is largely responsible for the material's elasticity and flexibility.^140,141 The hydrophobic nature elastin serves as a significant driving force in its self-assembly and aggregation.¹⁴² Here, the well-characterized elastin repeats in the hexapeptide Val-Gly-Val-Ala-Pro-Gly and pentapeptide Val-Pro-Gly-Val-Gly are chemically cross-linked by the hydrophilic domain.¹⁴³ The cross-linked peptides then supramolecularly organize into a nanoscale fibrillar structures that orient in the direction of the tissue applied load.¹⁴² Work on elastin-like synthetic sequences has been geared towards producing recombinant polypeptides that mimic both natural elastin's supramolecular assembly and its unique material properties.¹⁴² While mature cross-linked elastin is insoluble, synthetic peptides mimicking elastin's characteristic repeat have been designed to gain a greater understanding of the structure–function relationship of the protein. Such work is consistent with the formation of a β-turn structure around the Pro-Gly doublet, and an overall β-spiral secondary structure.¹⁴⁴ In 2002 Conticello developed a recombinant elastin-mimetic polypeptide [(VPGVG)₄(VPGKG)]₃₉ that exhibited mechanical properties similar to that of natural elastin, as well as its characteristic filamentous morphology.^145–147 When crosslinking reagent N-hydroxysuccimide was added, a reversible, temperature dependent hydrogel formed, presumably by an intermolecular condensation reaction between Lys residues and N-hydroxysuccimide substrates.¹³⁸ Work between Sallach et al. and Wu et al. described the design of a number of BAB recombinant co-block systems containing elastin-mimetic sequences.^26,148 In these systems, hydrophobic, elastin-mimetic polypeptide flanking sequences are separated by a central hydrophilic block. The elastin-mimetic domains are characterized by a repeating Val-Pro-Xxx-Yyy-Gly pentapeptide, while the central elastomeric domain is hydrophilic due to the presence of charged residues.¹⁴⁸ This co-block peptide can further aggregate, mimicking natural elastin's supramolecular structure while retaining its unique material functionality. Specifically, hydrophobic packing of the flanking domains in aqueous solvent drives self-assembly, while the central domain retains conformational flexibility and elasticity due to the presence of polar amino acid residues.¹⁴⁸ In contrast to chemical crosslinking between fibers as described by Wright and Conticello's 2002 study, this design utilized physical crosslinking to self-assemble, and demonstrated a number of advantages, including reversible self-assembly and the ability to control mechanical properties.^26,138 Further work reported similar elastin-mimetic polymers that combined both physical and chemical cross-linking into a single design.²⁶ In doing so, they were able to exert precise control over a variety of material properties unique to physical and chemical cross-linked systems. Le et al. demonstrated the design of a series of beaded fiber-forming elastin-mimetic double-hydrophobic block peptides establishing examples of next generation systems.²⁵ The reader is directed to the following reviews for more details.^29,149–151 These synthetic elastin matrices, oftentimes combined with synthetic collagen matrices, have been used to recapitulate features of the ECM in applications for tissue repair,^152–156 drug delivery,^29,157,158 and materials with tunable material properties.^{150,154,159–161}

Conclusion

Bottom-up engineering of peptide-based supramolecular structures has allowed biomimicry at multiple length scales, including nanofibrous morphologies that mimic native ECM. Learning from natural self-assembly cues, bioinspired scientists can now generate a series of materials that can closely recapitulate higher order protein structure by tailoring primary and secondary composition to replace dependence on synthetic or animal derived matrices that may cause adverse host reactions.

Authors' contributions

All authors participated in the preparation and review of the manuscript; VAK, BKW, and SMK wrote the manuscript.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: VAK has stock options in NangioTx, Inc which aims to translate some of the technologies presented in this manuscript towards clinical trials. No other authors report that they have no competing interests with the current work.