CHAPTER 1: Design and Synthesis of Peptides for Developing Biomaterials
Published:18 Nov 2020
In this chapter, an intelligent design of synthetic peptides and their implementation in biomaterials is discussed. Potential biocompatibility, bioactivity and availability through well-established synthetic methods indisputably make peptides one of the indispensable tools in developing new materials for biomaterials engineering. Synthetic peptides are used to display bioactivity by various approaches, such as supramolecular self-assembly forming biomimetic systems, preparation of peptide-based polymeric scaffolds, as well as post-synthetic immobilization on polymeric scaffolds. The design of the peptides is the most crucial part, as it defines functionality and the mode of bioactivity of the final product. A variety of design principles and fabrication of functional materials in peptide biomaterials are presented in detail in this chapter.
Synthetic peptides and their various derivatives are unarguably highly potent building blocks for the development of biomaterials. This is attributable to their close relation to proteins, potential biocompatibility, and ease of synthesis. There are mainly three approaches for employing peptides in the preparation of biomaterials: supramolecular self-assembly, peptide–polymer copolymerization, and post-synthetic covalent/non-covalent polymer modification.1,2 Each of these strategies requires its own peptide synthesis design with consideration of synthesis protocol and intermolecular and intramolecular interactions. Peptide-based supramolecular biomaterials are among the self-assembled functional materials that attract most attention from the scientific community. Programmed self-assembly of designed building blocks is important, primarily due to the resemblance to natural processes in living organisms, as well as the precision of structural elements involved in supramolecular organization.
1.2 Peptide Design
The most challenging issue in employing peptides in biomaterial development is the design of self-assembling peptides, because it requires information encoding at several levels of complexity, from basic non-covalent intermolecular interactions to the highest level of hierarchical organization. Practically, one ends up with intelligent design strategies, which in turn dictate programmable self‐assembly. This chapter mainly focuses on self-assembled peptide functional materials and their current or potential implementation in biomaterial development. Besides the self-assembly approach, development of peptide–polymer hybrids and post-synthetic modification of polymers with peptides also attract considerable interest. Among these is preparation of peptide–polymer block copolymers, peptide monomer-based polymers, and non-covalently modified polymers.
1.2.1 Self-assembling Peptides
Peptide amphiphiles (PAs) are among the most studied self-assembling amphipathic molecular entities of peptidic nature. A range of PA designs utilizing aliphatic, aromatic, or polymeric tail groups have emerged. Amphiphilic peptides are formed by an oligopeptide sequence and a covalently attached aliphatic molecule, which is typically 12–16 carbon atoms. Supramolecular ensembles of PAs range from nanofibers and nanoribbons to micelles and vesicles (Figure 1.1).3,4
One of the first bioactive peptide amphiphile system was used for biomineralization application by mimicking the structural and bioactive properties of collagen fibers.5,6 The effects of the peptide sequence and the type of aliphatic and hydrophobic residues on the morphological and structural characteristics were investigated widely. Typically, a minimum of a ten-carbon long aliphatic residue is necessary to induce the hydrophobic effect to cause unidirectional self-assembly. The changes in the peptide sequence have effects over bioactivity, in addition to non-covalent interactions resulting in self-assembled systems. The amino acids closer to the aliphatic segment of the peptide amphiphile molecule were found to result in spherical micelles, due to distressing the hydrogen bonding capacity,6 and flat tape-like structures were formed when alternating hydrophobic and hydrophilic amino acids were used.7 A variety of peptide epitopes are conjugated to the self-assembling molecules forming nanostructures.8–11 Systems displaying diverse bioactive epitopes, such as RGDS,12,13 IKVAV,9,14,15 FGF-2,16 and VEGF,8 have been developed successfully. Building on this concept PA-based biomaterials with regenerative function were developed. Bioactive PA nanofiber gels were shown to accelerate wound healing in burn injury (Figure 1.2a) (palmitoyl-VVVAAAEEEGGGGGRGDS-Am + palmitoyl-VVVAAAEEE-Am, lauryl-VVAGEGDK(p-sulfobenzoyl)S-Am + lauryl-VVAGK-Am),17,18 serve as nanofiber neurografts with neuroregenerative capability in facial nerve repair (palmitoyl-VVVAAAEEE-Am) (Figure 1.2b),19 facilitate regeneration of cartilage-like intervertebral disc tissue (lauryl-VVAGKPOG-Am + lauryl-VVAGE),20 induce cartilage regeneration (lauryl-VVAGKS(β-Glc)-Am + lauryl-VVAGE),21 and repair cardiac tissue defect after myocardial infarction.22
The PA hydrogel-based biomaterials are used to direct differentiation and transdifferentiation of cells. The co-assembled nanofiber hydrogel of lauryl-VVAGKKGHAV-Am and lauryl-VVAGE presenting the N-cadherin mimetic motif HAV was demonstrated to support the differentiation of mesenchymal stem cells into chondrocytes by promoting their commitment to the chondrogenic lineage.23 Co-assembled three-dimensional (3D) hydrogel of lauryl-VFDNFVLKK-Am and lauryl-VVAGEE PAs, with the tenascin-C-derived epitope VFDNFVLK on the surface, was observed to induce neuronal differentiation of PC12 cells.24 A three‐dimensional laminin-mimetic nanofiber hydrogel of lauryl-VVAGKKIKVAV-Am and lauryl-VVAGEE-Am PAs was also used for in vitro neural differentiation of PC-12 cells.25 Hydrogels based on combinations of lauryl-VVAGKS(β-Glc)-Am, lauryl-VVAGEK(p-sulfobenzoyl)-Am, and lauryl-VVAGE-Am PAs were used to direct differentiation of rat mesenchymal stem cells towards mesodermal lineages. The effects of glucose, carboxylate, and sulfonate groups presented on the peptide nanofibers were investigated by considering the variations in the differentiation profiles. The results showed that a higher sulfonate-to-glucose ratio was associated with adipogenic differentiation, and a higher carboxylate-to-glucose ratio was associated with osteochondrogenic differentiation.26 A supramolecular bioactive material based on a PA, palmitoyl-V2A2E4GIKVAV-Am, displaying a laminin-mimetic IKVAV sequence was used to induce neural transdifferentiation of human bone marrow mesenchymal stem cells. The PA molecules self-assemble into a supramolecular nanofiber hydrogel that induces neuroectodermal lineage commitment.27
The in vivo use of self-assembling PA,palmitoyl-VVAAEEEADEGVFDNFVLK-Am, carrying a tenascin-C signal (ADEGVFDNFVLK) for the redirection of endogenous neuroblasts in the rodent brain is reported. The PA, when co-assembled with palmitoyl-VVAAEE-Am, forms highly aligned nanofibers, displaying the migratory sequence of tenascin-C glycoprotein as an epitope. It was demonstrated that this injectable biomaterial is able to redirect migrating cells from the rostral migratory stream, evidenced by neuroblasts reaching the cortex instead of the olfactory bulb.28 The brain-derived neurotrophic factor (BDNF) mimetic sequence, cyclo(RKKADP), was incorporated into a supramolecular PA biomaterial capable of activating the BDNF receptor TrkB and downstream signaling in primary cortical neurons in vitro. The BDNF mimetic motif was only active when displayed on a peptide amphiphile supramolecular nanostructure.29
PA hydrogel-based biomaterials were also exploited as delivery tools. A hydrogel of palmitoyl-VVAAEE-Am was used to deliver sonic hedgehog protein, which promoted cavernous nerve regeneration and neurite formation in penile projecting neurons.30,31 Co-assembled nanofibers of palmitoyl-VVAAEEGDWFKAF-YDKVAEKFKEAF-Am and palmitoyl-VVAAEE-Am that display an apolipoprotein A1 mimetic sequence (DWFKAF-YDKVAEKFKEAF-Am) as the bioactive epitope were reported. This nanofibrous biomaterial, designed to target atherosclerotic plaque and enhance cholesterol efflux, was shown to encapsulate and deliver a liver X receptor agonist to increase efflux from murine macrophages in vitro.32 A co-assembled hydrogel of lauryl-VVAGEEE-Am and lauryl-VVAGKKK-Am PAs was used for local delivery of doxorubicin (Dox). This injectable supramolecular biomaterial did not show any considerable cytotoxicity and the encapsulation strategy enhanced the activity of the drug on cellular viability through prolonged release in vitro. Moreover, the local in vivo injection of the PA biomaterial with encapsulated Dox to the tumour site demonstrated the lowest tumour growth rate compared to direct Dox injection and increased the apoptosis of the tumour tissue cells.33
In addition to aliphatic molecules, aromatic groups are also conjugated to the peptides. Aromatic π–π stacking molecules, such as carbobenzyloxy, naphthalene, or fluorenylmethoxycarbonyl (Fmoc), were conjugated to peptides forming hydrogel materials. The first report of this type of self-assembling system dates back to 1995.34 Recently, peptide sequence has been shown to affect the structures of the self-assembled systems, such as spheres, fibers, tubes, and sheets, as shown in Figure 1.3.35,36 It is possible to include many functionalities, including bioactive groups37 and electroactive moieties, for a variety of applications.38 An Fmoc–FF peptide molecule was used to form antiparallel β-sheets based on spectroscopic and X-ray scattering analysis, while the aromatic Fmoc units form π-stacked aggregates.39 A similar structure was observed for Fmoc–LLL, but with a larger diameter.38 Fmoc-peptide systems have been investigated as a self-assembling building block based on aromatic short peptide derivatives for biomaterials.
To facilitate metabolomics analysis of stem cell differentiation, supramolecular hydrogels with tuneable stiffness were designed (Figure 1.4). In core–shell co-assembly of the peptide-based gelator Fmoc–FF and surfactant-like molecule Fmoc–S, the serine residue presented carboxylate functionality to the surface of the fibers,41 enabling subsequent cross-linking upon exposure to divalent cations (Ca2+) in tissue culture media. Formation of hydrogels with tuneable stiffness was achieved by varying the co-assembly concentrations. These gels facilitated stiffness-tuned stem cell differentiation without any biochemical functionalization. Developing bioactive materials for differentiation of stem cells into bone and cartilage formation is vital for new biomaterials and drug discovery.42
Biocompatible electroactive hydrogels based on co-assembly of oppositely charged tetra(aniline)-conjugated aromatic PAs, TA-VVAGEE-Am and TA-VVAGKK-Am, were used for neural differentiation of PC-12 cells. Improvement in neurite outgrowth was observed on the electroactive nanofiber hydrogel.43
A gemcitabine prodrug, integrated drug–carrier system based on co-assembly of Fmoc–EFFAAE-Am and Fmoc–KFFAAK-Am peptides into one-dimensional nanofibers was reported. Gemcitabine prodrug, which is gemcitabine covalently conjugated to the C terminus of Fmoc-protected glycine, was integrated into the peptide nanocarrier system through noncovalent interactions. The prodrug carrier system exhibited slow release and reduced the cellular viability of the 4T1 breast cancer cell line in a time- and concentration-dependent manner.44
Multidomain peptides (MDPs) are another class of self-assembling peptides that form nanofibrous hydrogels. The primary sequence design is based on a core of alternating hydrophilic and hydrophobic amino acids flanked by charged amino acids (Figure 1.5a), which results in the formation of nanofibers with a bilayer of β-sheets (Figure 1.5b). The addition of multivalent salts, such as those found in common buffers, promotes nanofiber elongation and cross-linking to form a viscoelastic hydrogel.45 The hydrogels are thixotropic and syringe injectable, which is useful for biomaterial localization in regenerative and therapeutic applications (Figure 1.5c).46,47
Ac-K2(SL)6K2-Am MDP hydrogel was used to heal full-thickness wounds in genetically diabetic mice; the hydrogel resulted in significantly accelerated wound closure with formation of thick granulation tissue, including dense vascularization, innervation, and hair follicle regeneration.48 In another study, hydrogel of the same MDP was observed to trigger angiogenesis and neurogenesis; the innate response to the material resulted in rapid cellular infiltration, production of a wide range of cytokines and growth factors, and finally transforming the synthetic biomaterial to a natural collagen-containing extracellular matrix.49
The previously reported highly angiogenic hydrogel of the MDP, Ac-K-(SL)3(RG)(SL)3-K-G-KLTWQELYQLKYKGI-Am,50 was also used as an engineered scaffold to mimic vascular endothelial growth factor (VEGF). In mice with induced hind limb ischemia, this injectable biomaterial promoted angiogenesis and ischemic tissue recovery.51
A hydrogel of MDP Ac-K(SL)3RG(SL)3KGRGDS-Am (SLac) was modelled ex vivo with intact dental pulp. Delivered centrally and peripherally within whole dental pulp tissue, the biomaterial was shown to be biocompatible and preserved local tissue architecture. In addition, odontoblast function and pulp vitality were sustained when the MDP scaffold was intercalated between dentin and the odontoblast region.52 MDP Ac-HAVDIGGKLDLKLDLKLDL with the N-cadherin mimetic motif (HAVDI) was co-assembled with Ac-KLDLKLDLKLDL in phosphate-buffered saline to form a hydrogel. Human mesenchymal stem cells encapsulated in the hydrogel demonstrated enhanced expression of chondrogenic marker genes and deposition of cartilage-specific extracellular matrix that was rich in proteoglycan and type II collagen.53
Recently, chemical functionality of hydrogels of Ac-K2(SL)6K2-Am, Ac-R2(SL)6R2-Am, Ac-E2(SL)6E2-Am, and Ac-D2(SL)6D2-Am MDPs was employed to govern the early host immune response (Figure 1.6). The bioactivity of ammonium, guanidinium, and carboxylate ions displayed on the nanofibers was studied in the context of the early inflammatory host response in a subcutaneous injection model. Although, all the studied peptide biomaterials possessed similar nanostructures and physical properties, they triggered considerably different inflammatory responses.54
MDP hydrogels were also used as delivery and localization tools providing spatiotemporal control over release. Orthogonal self-assembly of Ac-K(SL)3RG(SL)3KGRGDS-Am MDP molecules and growth-factor-loaded liposomes generated supramolecular composite hydrogels (MLCs). These composites acted as PIGF-1 delivery vehicles and, when injected subcutaneously in rats, showed spatial and temporal control of angiogenesis. These hydrogels showed high levels of cellular infiltration, creating a suitable environment for large stable microvasculature promoted by PlGF-1 release. The observed biological response was directly dependent on structural aspects of rationally designed MLCs developed to mimic the level of complexity seen in sophisticated natural systems.55 SLac was also used to obtain drug-triggered and ionically cross-linked self-assembled nanofibrous hydrogels. Using a multivalent phosphate, heparin, clodronate, trypan, and suramin ionic cross-linking strategy to create hydrogels for sustained long-term delivery of drugs was demonstrated.56 The same MDP was used in the development of a novel surgical haemostat based on snake-venom-derived batroxobin and self-assembled peptide hydrogel. This snake venom-loaded peptide hydrogel was applied via syringe and conformed to the wound site, resulting in haemostasis. This biomaterial demonstrated a facile method for surgical haemostasis, even in the presence of anticoagulant therapies.57 MDP hydrogel that capitalizes on cytokine delivery was used to tailor biological responses. This biomimetic material shows shear stress recovery and offers a nanofibrous matrix that sequesters cytokines. The biphasic pattern of cytokine release resulted in spatiotemporal activation of THP-1 monocytes and macrophages, with no pro-inflammatory environment observed.58
An injectable Ac-K2(SL)6K2-Am peptide hydrogel (STINGel) was developed to improve the efficacy of cyclic dinucleotide (CDN) immunotherapy. A novel biomaterial localized and provided controlled release of CDN, showing an eightfold slower release rate compared to a standard collagen hydrogel.59 By substituting one or two leucine residues in the middle of the same MDP with alanine residues, Ac-K2(SL)3SA(SL)2K2-Am and Ac-K2(SL)2(SA)2(SL)2K2-Am “missing tooth” variants were obtained, respectively. Through manipulation of the peptide primary sequence, a molecular cavity was incorporated into the hydrophobic core of the self-assembled MDP nanofibers, allowing for encapsulation and delivery of small-molecule drugs with poor water solubility. Hydrogels loaded with SN-38, diflunisal, and etodolac exhibited prolonged drug release profiles due to intrafibrillar drug encapsulation.60
1.2.2 Peptide–Polymer Hybrids
Traditional block copolymers can self-assemble in bulk due to microphase separation, or in selective solvents by formation of micelles, to form nanostructures.61 Peptides are molecules that can be tailored and they can be engineered to form a number of self-assembled structures due to their capacity to organize into α-helix or β-sheet conformations.62
The synthesis of a well-defined complex peptide–polymer enables control of the structural hierarchy of the block copolymers, for potential biomedical applications.63 The ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) are widely used to make polypeptides.64
Electroactive supramolecular polymers based on oligoalanine–oligoaniline–oligoalanine conjugates were reported. The electrochemically active oligoaniline blocks were functionalized with amines, which enabled the initiation of ROP of alanine N-carboxyanhydride (Ala-NCA). The tetra-aniline and hexa–aniline conjugates were prepared and the films of the supramolecular polymer promoted adhesion of fibroblasts and provided electrically triggered release of the clinically relevant anti-inflammatory drug dexamethasone phosphate.65
Poly(l-lactide-b-γ-benzyl glutamate) copolymers were synthesized by sequential ROP of l-lactide and the N-carboxyanhydride of γ-benzyl l-glutamate. The copolymers contained either crystalline poly-L-lactic acid (PLLA) or the liquid-crystalline columnar hexagonal morphology of poly-benzyl-l-glutamate (PBLG). Varying the temperature, reversible, local order−order transitions could be observed on these diblock copolymers.66 A novel class of tri-arm, star‐shaped, peptide-based block copolymers with self‐assembling nature and phase‐transition behaviour was synthesized by the combination of atom transfer radical polymerization (ATRP) and living ROP of the N‐carboxyanhydride of γ-benzyl l-glutamate.67 Similarly, by combining two living polymerizations, anionic and ring opening, multiblock multicomponent linear and miktoarm star (µ-star) peptide–polymer hybrids (macromolecular chimeras) were prepared using NCAs of γ-benzyl l-glutamate (BLG) and ε-tert-butyloxycarbonyl-l-lysine (BLL) (Figure 1.7).68
A series of novel antibacterial copolymers of polylysine and polycaprolactone (PCL), PCL16-b-Kn, demonstrating antimicrobial activity have been reported. The diblock copolymers were synthesized via ROP and facile block conjugation. The copolymers were shown to have low cytotoxicity and exhibit rapid bactericidal action against Staphylococcus aureus and Escherichia coli.69
The controlled polymerization of a peptide‐based monomer was reported by using a cyclic β‐sheet forming decapeptide gramicidin S, modified with a methacrylate handle. It polymerized via ATRP to produce a well‐defined gramicidin S‐containing polymeric system. The secondary structure of the peptide moiety was preserved in the polymeric material. This is an important example of the use of ATRP to create a synthetic polymer with a cyclic peptide as a side chain.70
A versatile methodology to prepare hybrid biomaterials by ATRP from resin-supported peptides has been established. As an example, GRGDS-functionalized poly(hydroxyethyl methacrylate) (pHEMA) was synthesized. Cell adhesion experiments showed that the GRGDS sequence promoted cell adhesion, in contrast to unfunctionalized pHEMA. By incorporating cell-signaling moieties in materials with defined molecular architecture, it is possible to control the interactions between polymeric materials and biological systems.71
Peptide–PCL–peptide conjugate was synthesized with the cell-binding RGDS motif and 3D printed into scaffolds (Figure 1.8). The peptide was additionally modified with the bio-orthogonal biotin moiety for visualization of peptide concentration and location, through fluorophore labeling. Peptide concentration on the scaffold surface increased with increasing concentration of the conjugate in the bioink; scaffolds printed with the highest conjugate concentration showed a substantial increase in NIH3T3 fibroblast adhesion.72
1.2.3 Peptides as Modifiers
Collagen mimetic peptides (CMPs) were effectively used to modify poly(ethylene glycol) (PEG)-based scaffolds with biosignals via triple helical peptide assembly. Photocross-linked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying CMPs were noncovalently modified with bioactive molecules via CMP–CMP triple helix hybridization. Designed peptides, (POG)8-G3-RGDSY-Am (CMP-RGD) and (POG)8-G3-KLTWQELYQLKYKG-Am (CMP-QK), were used to introduce cell-binding and pro-angiogenic sequences, respectively.73,74 It was also shown that the high binding affinity of Ac-KLTWQELYQLKYKGI-G3-(POG)9-Am (QK-CMP) to denatured collagens75 can be utilized to deliver angiogenic signals to scaffolds composed of heat-denatured collagens (gelatins).76
Another noncovalent approach was used to immobilize a bioactive peptide on an alginate matrix displaying covalently attached β-cyclodextrin (β-CD) via host–guest chemistry. Inclusion complex formation between molecular host (β-CD) and guests, such as 1-adamantane-amide-G5-RGDS (ada-RGDS), 1-naphthalene-amide-G5-RGDS (naphthyl-RGDS), and 1-adamantane-amide-G5-RGES (ada-RGES), was used to obtain biomaterials with dynamic display of bioactivity.77 The same strategy was used to decorate electrospun cyclodextrin nanofibers (CDNFs) with adamantane-conjugated bioactive peptides.78,79 A peptide, Ada-Ahx-GGKIKVAV-Am, displaying laminin-derived bioactive IKVAV epitope was noncovalently immobilized on the aligned CDNF scaffold (Figure 1.9). This non-covalent functionalization approach allowed fabrication of implantable scaffolds for peripheral nerve regeneration. While electrospun CDNFs introduced a three-dimensional biocompatible microenvironment to promote cellular viability and adhesion, the bioactive epitopes displayed on the surface guided the cellular differentiation of PC-12 cells.79
Recently, pHEMA cryogels with copolymerized β-CD units were prepared as an inert macroporous scaffold and non-covalently modified with adamantylated Ada-Ahx-GGRGD and Ada-Ahx-GGGHK peptides (Figure 1.10). A well-defined mitogenic effect of the immobilized peptides toward 3T3 and PC-12 cells was revealed. The synergistic action of RGD and GHK peptides induced a profound change in cell behaviour and morphology, which was attributed to a growth factor-like activity of the composition. The results established an effective procedure for the preparation of CD-modified pHEMA cryogels and their uniform in situ functionalization with bioactive peptide(s) of interest, and an informative study of cellular responses in the functionalized scaffolds.80
Supramolecular polymerization of peptides and their various derivatives is a promising approach in the development of biomaterials for tissue regeneration, drug delivery, and even drug discovery. Peptide-based extracellular matrix mimetics have proved to be potent scaffolds for cell differentiation/transdifferentiation in tissue engineering applications. Peptide-based hydrogels, owing to their potential to encapsulate both hydrophilic and hydrophobic drugs, are capable of establishing spatiotemporal control over release and providing enhanced therapeutic effects. In addition, biocompatibility, low cytotoxicity, and the biorelevant nature of peptide-based biomaterials render them superior to other alternatives.
R.G. acknowledges RFBR grant no. 19-03-01010.