Mini-review Received: 15 September 2013
Revised: 28 November 2013
Accepted article published: 5 December 2013
Published online in Wiley Online Library: 15 January 2014
(wileyonlinelibrary.com) DOI 10.1002/pi.4670
Biomimetic protein-based elastomeric hydrogels for biomedical applications Jasmin Whittaker, Rajkamal Balu, Namita R. Choudhury∗ and Naba K. Dutta∗ Abstract In recent years, protein-based elastomeric hydrogels have gained increased research interest in biomedical applications for their remarkable self-assembly behaviour, tunable 3D porous structure, high resilience (elasticity), fatigue lifetime (durability), water uptake, excellent biocompatibility and biological activity. The proteins and polypeptides can be derived naturally (animal or insect sources) or by recombinant (bacterial expression) routes and can be crosslinked via physical or chemical approaches to obtain elastomeric hydrogels. Here we review and present the recent accomplishments in the synthesis, fabrication and biomedical applications of protein-based elastomeric hydrogels such as elastin, resilin, flagelliform spider silk and their derivatives. c 2013 Society of Chemical Industry Keywords: biomimetic proteins; elastomers; hydrogels; tissue engineering; regenerative medicine
INTRODUCTION
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component of arterial blood vessels and artery wall), fibrillin, spider silks), byssus and abductin from bivalve molluscs, resilin from arthropods, gluten from wheat, frog glue, and artificial proteins.9,10,15 – 18 Such elastic proteins attract significant research interest due to (i) their biological and medical significance, particularly in human disease, and (ii) their unusual properties that provide opportunities to develop novel materials. They can be designed with properties and functions that go beyond those of known proteins, once the rules for tuning their properties have been established. These elastomeric proteins can be defined by their behaviour in terms of secondary and tertiary protein structure, that can be highly ordered or intrinsically disordered.19 Intrinsically disordered proteins (IDPs) such as elastin, resilin and flagelliform spider silk have gained increasing importance in the development of protein-based hydrogels as they display remarkable elasticity, stimuli-responsiveness and fatigue characteristics which can be harnessed for a range of applications. In this review, we discuss the recombinant synthesis, molecular structure, crosslinking, properties and development of the IDP elastomers elastin, resilin and flagelliform spider silk and their derivatives into hydrogel structures for use in biomedical applications (Fig. 1). Elastin is one of the main elastomeric insoluble proteins found in the natural ECM of animals that provides elasticity to different tissues and organs such as blood vessels, skin and lungs.12,13 Several elastin-like polypeptides (ELPs), comprising the consensus repeats of native elastin, that mimic the inherent elasticity
∗
Correspondence to: Namita R. Choudhury and Naba K. Dutta, Ian Wark Research Institute, Mawson Lakes Campus, University of South Australia, Mawson Lakes, Adelaide, SA 5095, Australia. E-mail:
[email protected],
[email protected] Ian Wark Research Institute, Mawson Lakes Campus, University of South Australia, Mawson Lakes, Adelaide, SA 5095, Australia
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Hydrogels are a class of three-dimensional crosslinked synthetic or biopolymer networks that are insoluble and capture large amount of water.1 – 3 Synthetic hydrogels are often unable to mimic the biophysical microenvironment of the extracellular matrix (ECM) that is required for biomedical applications, particularly tissue engineering.4 Moreover, tunability is critically important for the next-generation functional biomaterial’s application. Therefore, tunable polymers, biopolymers and their hydrogels have emerged as one of the most promising classes of soft condensed matter due to their potential in biotechnology, medicine, tissue engineering and controlled drug delivery applications. Designer biomimetic proteins synthesised through recombinant DNA techniques are an important class of such tunable protein-polymers. The structure and function of these protein-polymers can be tailored and controlled much more accurately than through synthetic techniques.5 In addition, the 3D pore sizes and the mechanical properties of these hydrogels may be tuned further through the use of different materials and the nature of the crosslinking interactions employed.1,6 In particular, biomimetic hydrogels derived from crosslinked elastomeric proteins are highly suitable for biomedical applications as they possess remarkable rubber-like elasticity, high resilience when stretched, large extensibility before rupture, outstanding resilience (reversible deformation without loss of energy) and excellent durability.7 – 9 In nature, many elastomeric proteins occur in a wide range of biological systems, where they have evolved to fulfil precise biological roles. They possess outstanding elasticity and play a number of different roles and are critical to the function of biological machinery. The different roles range from allowing for the jumping and flying ability of arthropods and the capture function of spider webs, to expansion and contraction of blood vessels and lungs in animals.10 – 14 Only a limited number of protein- and polypeptide-based elastomers have been demonstrated in the literature. These include proteins in vertebrate muscles and connective tissues (titin, elastin (the primary elastic
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most elastic and extensible of all the silks, is produced by orb weaver spiders and is important for the capturing function of spider webs.28 – 30 Studies into biotechnological approaches for developing proteins and polypeptides based on flagelliform spider silk have prompted significant research interest recently.29
Jasmin Whittaker received her BEng (Hons) in 2012 from the University of Adelaide, Australia. Currently, she is completing her PhD degree at the Ian Wark Research Institute, University of South Australia. Her PhD is related to the use of elastomeric biopolymers for biomedical applications
BIOMIMETIC SYNTHESIS OF ELASTOMERIC PROTEINS
Mr Rajkamal Balu is currently a PhD student at Ian Wark Research Institute, University of South Australia (UniSA). He is a recipient of an International President Scholarship (IPS) award at UniSA. He received his BTech (Industrial Biotechnology) degree from Anna University, India (2006), and his MTech (Nanotechnology) degree from SASTRA University, India (2008). Professor Namita Roy Choudhury is a Research Professor in Polymer Science at the Ian Wark Research Institute, University of South Australia. She received her PhD from IIT, Kharagpur, and subsequently did her postdoctoral research at CNRS, Mulhouse, France. Choudhury’s research interest spans from hybrid polymers to biomimetic polymers and tissue-engineering scaffolds. Professor Naba Dutta is a Research Professor in Polymer Interfaces and Nanomaterials Science at the Ian Wark Research Institute, University of South Australia. Professor Dutta has been involved in research in the areas of polymer nanomaterials for more than 22 years after finishing his PhD in 1991. A particular emphasis of his research is on multi-responsive polymers and their heterostructures of controlled composition, morphology, interfaces and functionality.
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of natural elastin have been synthesised using recombinant techniques.20 Resilin is an insoluble crosslinked structural protein commonly found in specialised regions of the body of most insects where there are highly repetitive movements. It has long been recognised for its outstanding elasticity and has been shown to play a significant role in the jumping, flying of dragonflies and fleas, and the sound production mechanisms in arthropods.21 It was first discovered by Weis-Fogh in locust wing-hinges and is currently identified as the most efficient elastic protein known.22 – 26 In recent years, resilin-mimetic modular proteins (RLPs) developed through recombinant methods have also evolved as potential biomaterial substitutes for a variety of applications including tissue engineering scaffolds.27 Finally, spiders are capable of producing a number of different silks that are among the most outstanding biomaterials in terms of their mechanical properties. Flagelliform spider silk, the
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The limited choice of polymers that self-assemble to form supramolecular complexes, along with challenges in their design and synthesis, has led to a modular protein engineering approach as a new direction to develop smart materials. This recombinant protein synthesis approach takes advantage of a diverse library of peptide domains and templates to synthesise protein-polymers with exquisite control over their properties and functions.31 – 33 The first step in the recombinant synthesis process involves the development of the desirable gene sequence. Following this, the sequences are inserted into the plasmid vector and cloned into the host cells (commonly Escherichia coli), followed by expression, harvesting and purification (Fig. 2).29,33 Here, we briefly discuss the synthesis of elastomeric proteins derived from the sequences of elastin, resilin and flagelliform spider silk. Once the proteins have been synthesised, the development into elastomeric hydrogels is possible, which will be discussed further in the crosslinking and fabrication sections of this review. The gene sequence identification of elastin from human origin (GenBank AAC98394), resilin from Drosophila melanogaster (GenBank AAF57953) and the flagelliform silk protein from Nephila clavipes (GenBank AAC38846) opened new routes to engineering ELPs, RLPs and recombinant flagelliform silks (FLP) with properties comparable to those of natural proteins.20,34 – 36 Recombinant expression of proteins in E. coli has been employed to design and synthesise many ELPs with different functionalities that display advanced bio-functional behaviour and allow for different crosslinking methods.37 – 42 These ELPS have been developed into a range of biomaterials through different fabrication methods that will be discussed further in later sections. Elvin et al. synthesised the first recombinant elastomeric resilin, rec1-resilin, through expression of the first exon of the Drosophila CG15920 gene in E. coli.43 This was followed by the development of several other RLPs including An16, Dros16, RLP12 etc.26,44 – 46 These RLPs have been further developed through recombinant techniques to include various bio-functional domains.47 – 49 The fabrication of the aforementioned RLPs with respect to biomedical applications will be further discussed in the following sections. Studies into biotechnological approaches for developing proteins and polypeptides based on flagelliform spider silk have been initiated in recent years. This has led to some initial work using flagelliform silk motifs for the fabrication of hydrogels for biomedical applications.28,29,36,50 The need to use recombinant methods for FLPs is critical due to the cannibalistic nature of spiders and the difficulty in collecting the flagelliform silk from the spinnerets on the spider.28,29 The development of methods for synthesising constructs based on the flagelliform silk protein recently has led to opportunities for fabrication of this material for biomedical applications, which will be discussed in more detail in the next sections.
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Biomimetic protein-based elastomeric hydrogels
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Figure 1. Scope of the review: development of elastomeric proteins into hydrogels for use in biomedical applications.
Figure 2. Schematic representation of key steps involved in the synthesis of recombinant proteins.
STRUCTURAL FEATURES OF PROTEIN-BASED ELASTOMERIC HYDROGELS
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Structural requirements and mechanism of elasticity Rubber-like elasticity in elastic proteins is characterised by their ability to undergo large deformation without rupture. By doing so, the protein efficiently stores the energy of deformation and subsequently returns to its original state on unloading of the deformation or stress. The mechanism to describe the elasticity of structurally disordered elastomeric proteins, such as elastin, resilin
and flagelliform silk, is attributed to entropy.19 On stretching a disordered polymer, the chain entropy is reduced, and once this strain is released the high entropy condition is restored.19,51 From a structural perspective there are two criteria that must be fulfilled to impart rubber-like elasticity in the elastomers.10 First, the individual units must be conformationally flexible in order to respond rapidly to an external force.8,10,19 In addition, the units must form a network through crosslinking interactions (physical or chemical).8,10,19 The crosslinks are required to distribute the imposed stress uniformly throughout the system and to prevent the separation of polymer chains when force is applied−which would destroy the structural system (Fig. 3).19,52 Essentially, elastomeric proteins must exist in a hydrated or polar solvent environment in order to act in a rubberlike way.9,14,24 In the dry state, intermolecular interactions (even in the elastic proteins) are strong and the glass transitions T g s (cooperative segmental motions) are only possible at high temperatures (T g room temperature), just prior to chemical decomposition. In hydrogels, water and/or polar solvents act as plasticisers by providing alternative, mobile hydrogen bond donors and acceptors for peptide groups. This results in a decreased energy barrier between different conformational states, allowing T g to occur at lower temperatures (T g room temperature). Thus the internal chain
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Figure 3. Schematic diagram showing the mechanism of elasticity for crosslinked proteins (Reprinted with permission from reference 51).
dynamics and interactions of the proteins with water and/or polar solvents are normally responsible for the elasticity and resilience of the proteins.5,13 Thus, the protein or polypeptide chains must form a crosslinked hydrogel-type structure in order to possess the remarkable elastic properties. The aforementioned requirements are satisfied by the presence of two different types of domains that can be characterised as either elastic repeating sequences or crosslinking domains.8 A minimum of one of each type is present in the structure of an elastic protein, and the lengths of the elastic domains and extent of crosslinking govern the overall elasticity of the material. In the following section the structural features of the elastin, resilin and flagelliform silk proteins are highlighted.
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Primary and secondary structure of elastomeric proteins Elastin, resilin and flagelliform spider silk are IDPs containing a large proportion of unordered structures (Figure 4), which are thought to be responsible for their remarkable flexibility and elasticity.14,53 – 60 Figure 5 gives a schematic representation of the primary structure of proteins in general and the types of secondary structures that are present in elastin, resilin and flagelliform silk proteins. Table 1 summarises the structural features of these IDPs in their native form. From Table 1 it can be seen that all three proteins have a large proportion of proline and glycine in their structures. The content of glycine and proline in elastomeric proteins is thought to be linked to the observed elastic behaviour.61 Proline and
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glycine are known as ‘helix breakers’ because they disrupt the regularity of the α-helical backbone conformation and are commonly found in turns. Moreover, the unique property of natively unfolded protein sequences is a combination of low overall hydrophobicity and large net charge.62 The mean hydrophobicity and mean net charge comparison of the native forms of elastin, resilin and flagelliform silk protein with hydrophilicity is shown in Fig. 4(A). Figure 4(B) suggests the naturally disordered regions of the proteins by the PONDR® (Predictor of Naturally Disordered Regions) algorithm.63 From these predictions, all three proteins are largely disordered in structure. Figure 4(C) shows the hydrophobicity of proteins through the hydropathy plot.64 Elastin is shown to be relatively hydrophobic, with resilin largely hydrophilic and flagelliform silk largely hydrophobic with some hydrophilic segments. The secondary structures of these elastic proteins predicted by discrimination of secondary structure class routine (DSC) is shown in Fig. 4(D).65 As expected, for each of these proteins a large proportion of the random coil structure is predicted, with some β-strand, which is in agreement with experimental techniques (Table 1). In addition, the polyproline II helical (PPII) conformation has also been found to exist in both elastin and resilin. The PPII conformation is highly flexible and has the ability to convert reversibly between β-strand and β-turn structures (Fig. 5).66 This conformational flexibility through inter-conversion between secondary structural types is a requirement for elastic properties, as discussed above. Furthermore, it has recently been found that some proportion of β-sheet in the flagelliform silk spacer region that occurs between repetitive sequences may contribute to the strength of this material; however, the protein is predominantly disordered.29,67 Therefore, it can be seen that elastin, resilin and flagelliform silk possess a number of common features. These include repeating units that make up the structure, a largely unordered structure with the possibility of inter-conversion between β-strand and PPII conformations, and a large proportion of glycine and proline in the amino acid sequence.7,10,53 However, resilin differs significantly in the fact that the overall structure is hydrophilic, whereas elastin and flagelliform silk are predominantly hydrophobic.53 These observations are very interesting and are reported to be related to the elastic protein’s biological roles and mechanical properties.11 The different structures of these elastomeric proteins lead to a range of different properties, including stimuli-responsiveness, that can be harnessed for a wide range of industrial and biomedical applications. The importance of the development of entropic elastic force and the occurrence of hydrophobic association to display rubberlike elasticity in ELPs has been discussed in detail by Urry and co-workers.68,69 Recently, Qin et al. elaborated on the fundamental role of β-turn-related structures in the elasticity of RLPs.70 Crosslinking of elastomeric proteins to form elastomeric hydrogels Biomimetic proteins based on elastin, resilin and flagelliform silk can be physically or chemically crosslinked to form elastomeric hydrogels. Soluble elastins can be crosslinked between lysine residues in alanine-rich regions to form insoluble elastomeric polymers. These crosslinks may be formed through γ -irradiation and chemical/enzymatic crosslinking procedures.71,72 In flagelliform silk analogues, the incorporated lysine groups in the glycine-rich regions provide crosslinkable sites. The post-translational fabrication of an elastomeric matrix by physical crosslinking (hydrogen and hydrophobic bonds) and the formation of secondary protein structures are responsible for the formation of protein-based gels.14,29,67 Resilin does not have a specific crosslinking domain,
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Table 1. Common structural features of elastomeric proteins Protein-based elastomers Elastin
Glycine content (mol%) 29
Proline content (mol%) 12.4
Resilin
34.2
10.3
Flagelliform silk
46.8
13
Nature of crosslinking Crosslinks between lysine residues in alanine-rich regions
No particular crosslinking domains Di- and tri-tyrosine crosslinks Non-covalent crosslinks
Secondary structure Repetitive domain β-strands β-turns PPII Crosslinking domain α-helix PPII β-turns Random coil β-turn conformations Random coil β-sheet (present in spacer regions)
Reference 6,7,19,66
19,43,53,73,76
19,29,67,77,78
Figure 4. (A) The mean hydrophobicity versus net charge plot. The solid line represents the calculated border between folded (above) and natively unfolded (below) proteins. (B) Prediction plot of naturally disordered regions by the PONDR® algorithm. If a residue value exceeds a threshold of 0.5, the residue is considered disordered. (C) Kyte−Doolittle hydropathy plot of proteins. A negative overall score predicts the hydrophilicity of the protein. (D) Secondary structure prediction by DSC.
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PROPERTIES OF ELASTOMERIC HYDROGELS Mechanical properties The elastomeric capability of a material is one of the most important characteristics to consider for the development of structures for a range of biomedical applications. Elastic properties are important for regenerative medical applications as many of the tissues found in the human body have elastomeric character and must be resilient upon stretching. A comparison of the mechanical properties of elastin, resilin and flagelliform spider silk is shown in Fig. 6(A).11 As shown, all three proteins are highly elastic, with flagelliform silk displaying improved toughness, which is thought to be due to the small amounts of β-sheet present in the spacer
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which is dissimilar from elastin and flagelliform spider silk. However, the tyrosine residues in the hydrophilic repeat sequences can participate in crosslinking (enzyme mediated crosslinking or photo-crosslinking) reactions to form di- and tri-tyrosine crosslinks.43,73,74 These crosslinks are important as they are responsible for the elasticity and also fluorescence exhibited by resilin.75 In addition, temperature- and pH-responsive behaviour can lead to physically crosslinked gels in appropriate conditions, which will be discussed in more detail later. However, for all the elastomeric hydrogels, there is a requirement for some type of crosslinking in order to impart the remarkable elastic properties.
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Figure 5. Schematic representation of the primary structure of a generic protein, with the amino acids represented by beads. A schematic representation of the secondary structures that contribute to the conformations of the proteins elastin, resilin and flagelliform spider silk. The cyclic rings that are a part of the proline amino acid structure are represented as rings in the PPII secondary structure.
Figure 6. (A) Stress–strain plot of elastin, resilin and flagelliform spider silk (Reprinted with permission from reference 11). (B) Creep behaviour of crosslinked rec1-resilin hydrogel at 25 ◦ C (Reprinted with permission from reference 80).
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regions.67 Elastin is important for the mechanical integrity of tissues in the body.79 With regard to the definition of elastomeric materials, elastin has been shown to possess a rubber-like elasticity with high resilience, low stiffness and large strain.79 Native elastin has been found to have a Young’s modulus of 300–600 kPa and an elongation at break of 100%−150%, which are dependent on the hydration level.9,20 With lower hydration levels elastin behaves as a rigid polymeric glass; however, it becomes rubbery at higher hydration levels.9 ELPs with comparable mechanical properties have been developed and crosslinked through a range of different methods (Table 2). The swelling ratio of gels can be related to the extent of crosslinking and mechanical properties of the gels (Table 2). Native resilin has been shown to have an extremely high resilience (97%), with a Young’s modulus of 50–300 kPa, ultimate tensile strength of 60–300 kPa and an elongation at break of 250%−300%.9 Qin et al. investigated the mechanical properties of full-length and crosslinked resilin using AFM.73 It was reported that the resilience of resilin exceeded that of highly resilient rubbers, with values of 94% ± 1% and 96% ± 2% for the full-length and crosslinked forms, respectively.73 Furthermore, in a similar way to elastin, resilin is hard and brittle when dehydrated, and swells in aqueous solution where it becomes rubbery and elastic.75
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In addition, like native resilin, resilin-mimetic protein-polymer rec1-resilin displays viscoelastic behaviour, and is classified as a relatively soft material as it swells with an 80% uptake of water at equilibrium.43 DSC studies of ruthenium mediated crosslinked rec1-resilin gels at different hydration levels indicated that the dehydrated gel was brittle with a glass transition temperature (T g ) of >180 ◦ C, which dramatically decreased with increasing hydration, and it exhibited rubber-like elasticity above a critical hydration level (Fig. 7(A)).80 Nano-indentation studies revealed that even with little hydration (