Protein-based functional nanomaterial design for bioengineering applications.

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Protein-based functional nanomaterial design for bioengineering applications Malav S. Desai1,2 and Seung-Wuk Lee1∗ In this review article, we describe recent progress in the field of protein-based bionanomaterial design with focus on the four well-characterized proteins: mammalian elastin and collagen, and insect-derived silk and resilin. These proteins are important structural components and understanding their physical and biochemical properties has allowed us to not only replicate them but also create novel smart materials. The ‘smart’ properties of a material include its ability to self-assemble, respond to stimuli, and/or promote cell interactions. Such properties can be attributed to unique structural modules from elastin, collagen, silk, and resilin as well as functional modules identified from other proteins directly or using display techniques such as phage display. Thus, the goal of this article is to not only emphasize the types of protein-based peptide modules and their uses but also encourage and inspire the reader to create new toolsets of smart polypeptides to overcome their challenges. © 2014 Wiley Periodicals, Inc. How to cite this article:

WIREs Nanomed Nanobiotechnol 2014. doi: 10.1002/wnan.1303

INTRODUCTION

P

roteins are an essential part of all biological systems. They have diverse and intricate life-sustaining functions and have evolved tremendously to make us all fit for survival. Among the numerous proteins that exist, structural proteins are of particular interest in the design of nanomaterials for bioengineering and medicine. By understanding and using the naturally occurring proteins as a starting point to design new materials, we can improve upon evolution and design solutions that currently do not exist. The proteins that will be discussed in this review are the mammalian elastin and collagen as well as insect-derived silk and resilin. These proteins have been studied extensively and have been used as scaffolds for tissue engineering and drug delivery.1 Protein-based polypeptide (PBP) materials possess ∗ Correspondence

to: [email protected]

1 Department

of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

2 Physical

Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Conflict of interest: The authors have declared no conflicts of interest for this article.

various advantageous features: (1) genetic engineering allows for the synthesis of precisely designed sequences, (2) recombinantly expressed PBPs enable preparation of monodispersed stock materials, and (3) careful sequence selection can facilitate mimicking natural extracellular matrix without the fear of rejection and disease transmission compared with allografts and xenografts or the intricate chemical synthesis involved in synthetic scaffolds (Figure 1). Additionally, PBPs are easy to use in biomedical applications owing to their innate biocompatibility and biodegradability.2,3 PBPs described here are relatively simple polypeptides composed of the identified consensus sequences that hold the major properties of their host protein such as elasticity of elastin and strength of silk. Recombinantly expressed PBPs can be either full protein genes, for instance, recombinant tropoelastin (rTE) and recombinant collagen, or the fully synthetic versions composed of tandemly repeated consensus sequences. The fully synthetic PBPs are of great interest as they allow for the incorporation of functionalities abnormal to their natural counterpart, for example, cell adhesive resilin4 and silk5 or hydroxyapatite (HAP)-binding elastin.6 The idea

© 2014 Wiley Periodicals, Inc.

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Design PBP with desired number and sequence order of modular domains

1. Modular PBP design: Structure domain 1

Structure domain 2

Functional domain 1

2. Genetic engineering:

Functional domain 2

Design and construct gene of interest

Construct plasmid and transform expression hosts E. coli

Insert gene into vector

Transform E. coli with plasmid Culture expression hosts and extract PBP to obtain a homogeneous stock material

3. Express and Purify PBP:

Purify PBP E. coli batch culture to express PBP

Homogeneous modularly designed PBP

FIGURE 1 | General approach to design protein-based materials: (1) Once the design parameters such as self-assembly, stimuli response, or specific mechanical properties are identified, primary sequence of the protein-based polypeptide (PBP) can be designed using different structural and functional modules. The gene of interest can be formulated and constructed based on the designed primary sequence. (2) Next step is to insert the gene of interest into a genetic vector. The resulting genetic construct can then be transformed into an expression host such as Escherichia coli . (3) PBP can be expressed and purified from a batch culture of the engineered E. coli to obtain a homogeneous population of stock PBP with the intended properties.

of incorporating functional modules into recombinant PBPs is nothing new; however, creating more comprehensive PBPs with multiple functionalities is a growing field.7 The development of more extracellular matrix-like PBPs is possible as now a single network of PBP can contain cell adhesion sites, biomolecule-binding sites, matrix metalloprotease sites, and so forth. Additionally, combining multiple structural motifs to create PBPs such as silk–elastin,8 silk–collagen,9 and even resilin–elastin–collagen10 has led to self-assembling novel materials with previously unobserved properties. In this review, we will first provide brief backgrounds for each of the four structural proteins followed by updates on the types of strategies being used for developing PBP-based tissue engineering and drug delivery materials. This is followed by a description of a range of modular PBPs developed for similar applications and future prospective on the field. Comprehensive collections of research for each PBPs such as elastin,1,2,11–15 collagen,1,15,16 silk,1,15,17–20 and resilin1,21,22 can be found in other recent reviews.

PROTEINS IN NANOMATERIAL DESIGN Elastin and Elastin-Like Polypeptides Elastin is one of the major mammalian structural proteins present in blood vessels, lung epithelium, skin, and other tissues to provide them with high elasticity and resilience.23 The highly repetitive elastin proteins are secreted by cells in their vicinity and cross-linked soon after. This cross-linked (or mature) elastin is fibrous and hydrophobic making it insoluble and difficult to isolate and sequence. To study elastin, it was critical to isolate the 72-kDa soluble protein known as tropoelastin upon secretion from cells.23,24 Tropoelastin is composed of lysine-rich hydrophilic cross-linking domains and elastic hydrophobic domains. Once outside the cell, lysine is enzymatically converted to allysine (a reactive moiety that forms desmosine or isodesmosine cross-links) by lysyl oxidase, a copper-dependent enzyme.24 As a result, soluble tropoelastin was first isolated from copper-deficient pigs by Sandberg et al.25,26 This was a major breakthrough in identifying the protein


WIREs Nanomedicine and Nanobiotechnology

(a)

Protein-based functional nanomaterial design

(Val-Pro-Gly-Val-Gly)n O N

H N

O

H N

H N N H O

O O

(b)

100 90

T < Tt

T > Tt

Turbidity (% of Max)

80 70 60 50 40 30 20 10 0 20

30 40 Temperature (°C)

50

FIGURE 2 | Elastin-like polypeptide sequence and stimuli response. (a) Consensus sequence derived from tropoelastin (Val-Pro-Gly-Val-Gly) can be tandemly repeated to synthesize elastin-like polypeptides (ELPs). (b) Stimuli-responsive properties of the ELPs, which exhibit inverse temperature transition. They are highly soluble in low temperature and coacervate above low critical solution temperature (T t ). (Reprinted with permission from Ref 27. Copyright 2013 American Chemical Society)

sequence and genes expressing this highly repetitive protein. Since then, there have been numerous studies about the biochemistry and structure of tropoelastin increasing our understanding of the role of elastin and its potential to solve problems. As mentioned earlier, tropoelastin is a highly repetitive protein with hydrophilic and hydrophobic domains. The hydrophilic domains mainly contain a series of lysine residues interspersed with alanine, while the hydrophobic domains contain repetitive sequence units such as the tetrapeptides, pentapeptides, and hexapeptides ‘VPGG’, ‘VPGVG’, and ‘VAPGVG’, respectively.24 The hydrophobic domains are the source of elasticity and the unique thermo-responsiveness of proteins related to elastin. These two properties are recaptured within recombinantly produced elastin-like polypeptides or ELPs (also known as elastin-like recombinamer, ELR) that commonly use the hydrophobic domain-derived pentapeptide repeats ‘VPGVG’ or more generally ‘VPGXG’ (‘X’ can be any amino acid except proline)2 (Figure 2). In mammalian tissues, the intriguing thermal response of tropoelastin is critical for mature elastin formation as the phenomenon localizes the

secreted tropoelastin until cross-linking. Localization occurs as tropoelastin phase separates into a coacervated state above its characteristic transition temperature (T t ). This phenomenon is fully reversible and is known as inverse temperature transition (ITT).24 Thus, ELPs also have ITT and numerous sequences produced over years of research show that this ITT depends on the sequence (i.e., hydrophilicity of the guest residue ‘X’) and the length/molecular weight (Mw ) of the ELP, as well as the pH and ionic strength of the environment.28,29 Understanding the mechanisms for the properties of elastin and its derivatives has been a challenge as the protein cannot be crystallized making structure determination and visualization challenging. Although only supported by circumstantial evidence, Urry has claimed from early on that elastin has a 𝛽-spiral structure with ‘VPGV’ sequences creating type II 𝛽-turns.30 Nonetheless, this is not probable because a rigid 𝛽-spiral structure with intrachain hydrogen bonding would contradict the rubber-like entropic mechanism observed for the elasticity of the protein; for example, stretching a single tropoelastin chain using an atomic force microscope does not produce a saw-tooth profile expected from unfolding of secondary or tertiary structures but instead shows single and repeatable change in cantilever deflection.31 Recent studies using complex two-dimensional nuclear magnetic resonance (2D NMR) modalities to determine distances between 13 C–1 H and 13 C–15 N as well as to observe 15 N within labeled elastin reveal the lack of a 𝛽-spiral structure but indicate that the entropically driven elasticity mechanisms rely on the entropy of water around hydrophobic elastin segments as opposed to the entropy of intrachain hydrogen bonding.32,33 This conclusion agrees with the tropoelastin chain stretching experiment in which elastin returns to its original conformation or something similar after the chain is relaxed. Additionally, it also helps explain the thermo-responsiveness of elastin that depends on the changes in the entropy of water molecules resulting from the changes in water/elastin hydrogen-bonding stability as the temperature increases. The theory can also be used to explain the increases in the proportions of ordered structures as seen in Fourier transform infrared (FTIR) and circular dichroism spectra resulting from elastin dehydration during its ITT that likely concentrates 𝛽-turns as the protein coacervates.34,35 In biomaterial research, three main derivatives of elastin have been used: 𝛼-elastin, recombinant human tropoelastin (rTE), and ELP (or ELR). The commercially available 𝛼-elastin is obtained from processed mature elastin derived from bovine tissues; however,



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these polypeptides have predefined sequences, a broad distribution of Mw , and can have batch-to-batch variability as they are tissue derived.36 rTE and ELP allow for better control of polypeptide dispersity with the fully synthetic ELP being the most biochemically customizable.2 Recombinant expression of elastin derivatives is mainly done in engineered Escherichia coli with relative ease of protein purification due to the thermo-responsiveness of elastins.2 As will be discussed later, elastins are extremely valuable for making highly extensible structures as well as for stimuli-responsive applications including stimuli-triggered self-assembly and molecule delivery. Outside of their usefulness as structural proteins, the thermo-responsive ELP sequences can also be used as fusion tags for purification of other recombinantly expressed proteins. Examples include purification of thioredoxin and tendamistat as shown by Meyer and Chilkoti, and more recently to purify beak and feather virus capsid proteins expressed in tobacco plants by Duvenage et al.37,38

Collagen and Collagen-Like Polypeptides Collagen is the most abundant structural protein in mammalian tissues. The characteristic structure of collagen is the triple helix composed of three separate 𝛼 chains that form homotrimers if the chains are identical or heterotrimers if they are different39 (Figure 3). In addition to the six 𝛼 chains, 𝛼1 to 𝛼6, further complexity arises as 𝛼 chains can be composed of different domains owing to alternative splicing of exons.39 Human collagen

Space-fill model

Triple -helix

Ball and Stick model

FIGURE 3 | Human collagen structures that are visualized using PDB: IBKV in two different forms: Space-fill model (left ) and ball and stick model (right ).

The variety of triple helices can then assemble into higher ordered supramolecular structures such as fibrils, beaded filaments, anchoring fibrils, networks, and hexagonal networks in addition to fibril-associated collagens with interrupted triple helices (FACITs) that latch onto assembled fibrils.39 So far, 29 different types of collagen have been identified serving different purposes in tissues.39,40 Among these, collagen type I is a major component forming tissues such as skin, tendons, blood vessels, organs, and bone.41,42 Collagen type III is also present in many tissues alongside collagen type I, while collagen type II makes up majority of the cartilage tissues as well as vitreous humour in the eye.41,42 Because of the relative abundance and applicability of these three types of collagen, they have been used in biomedical research as well as for more basic research to understand collagen structure and stability. The polypeptide chains of collagen are composed of highly repetitive tripeptide of the general form ‘GXY’.39,43 The presence of hydroxyproline (O) at ‘X’ and proline at ‘Y’ is relatively common in collagen sequences. Through studies of chemically synthesized collagen-mimetic peptides (CMPs) of the form [GOP]n , researchers have identified that hydroxyproline and proline are critical for the stability of mammalian collagen triple helices as the residues constrict the conformation of the chains to entropically stabilize the structure even at 37∘ C.16,44 The stereoelectronic effects of the electronegative hydroxyl group on the hydroxyproline have been indicated to promote the trans-isomer of the hydroxyprolyl amide bond, thus increasing triple helix stability.16,45 Although this knowledge is valuable, recombinant synthesis of collagen-like polypeptides (CLPs) still remains a challenge. Chemical synthesis allows the use of hydroxyproline, an unnatural amino acid; however, CMP/CLP is of limited length as well as expensive to produce in large quantities. Biological systems such as E. coli lack prolyl 4-hydroxylase required for post-translational modification of proline to hydroxyproline and the high G-C content of CLP genes also limits the size of peptide that can be successfully expressed. Other organisms such as plants and yeast have thus been commonly used to express CLP with the drawback of increased genetic complexity. A solution to this problem came to light upon the identification of collagen-like sequences in bacteria such as Streptococcus pyogenes, Bacillus anthracis, and Clostridium perfringens.46–48 This bacterial protein capable of forming triple helices is naturally expressed on the cell walls of the host bacteria and is used by the bacteria to anchor onto human cells.47 These proteins are also composed of repeating ‘GXY’ triplets with ‘X’ and ‘Y’ positions having




high frequencies of charged amino acids such as lysine, glutamic acid, aspartic acid, as well as rigid proline residues. The presence of charged residues enables bacterial collagen to mimic mammalian collagen-like triple helical structures without the need for hydroxyproline.48 This phenomenon has also been replicated using chemically synthesized CMPs containing charged amino acids instead of [GOP]n sequence.45 The bacterial collagens have been shown to have melting temperatures around 35–39∘ C,47,48 which is quite remarkable as it makes this simple and scalable recombinant CLP useful for biomedical applications. Currently, a critical property missing from bacterial collagen is that the protein cannot undergo mammalian collagen-like hierarchical assembly into complex structures.47 However, research using CMPs or model collagen-like particles to understand collagen stability and self-assembly is already underway and will help determine the criteria useful for future design and tuning of bacterial CLPs.45,49,50

Silk and Silk-Like Polypeptides Silk is one of the most important and ancient insect-derived protein materials used by humans. The protein has come a long way from being used in textiles to being FDA approved for use in various

biomedical applications.17 Silk is expressed by many insects as a supportive structural material and it has piqued the interest of many owing to its astonishing properties. It is extremely tough with high strength and elasticity, properties that tend to be mutually exclusive. In fact, silk can be compared to and has been shown to surpass the properties of materials such as tensile steel and kevlar.17 Among the different sources of silk, silkworm silk and spider silk have been studied heavily with dragline spider silk being the toughest of all types.17 Several domains and consensus repeat units have been identified in natural sequences of spider silk, for example, rigid 𝛽-sheet-forming ‘[A]n ’ and ‘[GA]n ’ repeats, highly elastic 𝛽-spiral-forming ‘GPGGX’ and ‘GPGQQ’ repeats, 310 helix-forming ‘GGX’ repeats, charged nonrepetitive spacers, and Nand C-terminal domains critical for pH-responsive fiber spinning in insect glands17,18,51 (Figure 4). The mechanical properties of silk depend on the order and proportions of crystalline and elastic domains involved, thus making the proteins intrinsically modular. Spiders alone combine these different modules in varying proportions to create an assortment of silks for example: dragline silk from major ampullate glands, vicid silk from flagelliform glands, and adhesive silks from aggregate and piriform glands as well as accessory silks such as tubiliform silk from

FIGURE 4 | Schematic showing the different types of silks spun by spiders. Each type of silk has different modular domains that vary the strength, elasticity, and adhesiveness of the silk. (Reprinted with permission from Ref 52. Copyright 2013 John Wiley and Sons)



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cylindrical glands and capture silk from aciniform glands.52,53 Among these proteins, the dragline silks, major ampullate spidroin 1 and 2, form the toughest fibers and are therefore used extensively for recombinant expression.52 Highly extensible silks have also been studied by recombinant expression of flagelliform silk containing the elastic ‘GPGGX’ repeats.54 Recently, a chimeric silk composed of a combination of major ampullate spidroin 2 and flagelliform silk modules was also created to show the formation of a highly extensible yet strong silk-like polypeptide (SLP) with a Young’s modulus of 4.6 GPa, toughness of 93.5 MJ/m,3 and extensibility of 80.3%.55 Although individually, the native dragline (10 GPa; 160 MJ/m3 ; 27–35%, respectively) and flagelliform (0.003 GPa; 150 MJ/m3 ; 200–270%, respectively) silks have higher toughness and excel in Young’s modulus and failure strain, respectively, the properties of the chimeric SLP are remarkable considering that it is only one fifth of the native protein length.55 Chimeric and multimeric SLPs combining multiple properties in a single polypeptide will be extremely valuable in the future especially if the hurdle of recombinant expression is overcome. SLP length is a critical factor defining its mechanical properties, but longer proteins are challenging to express owing to factors such as highly repetitive gene sequences rich in guanine–cytosine pairs, high glycine content, and insoluble expression products.56,57 Fortunately, over time, longer SLPs have been expressed in a variety of hosts such as bacteria (E. coli), transgenic silkworms, transgenic plants, and mammalian cells.57 Recently, Xia et al. successfully expressed a full-length 284.8-kDa SLP composed of major ampullate spidroin 1-derived sequence from Nephila clavipes using engineered E. coli.56 Compared to this, the more commonly expressed SLPs using similar sequences tend to be in the range of 60–130 kDa. In addition to the protein sequence and length, silk properties are also affected by the process of fiber spinning. As revealed through studies of spider silk glands, a highly concentrated aqueous silk dope undergoes phase separation and transitions to a solid from due to ion exchange, acidification, and water removal as it travels out from ampulla (sac) through a tapered duct.58 It is proposed that the amphiphilic silk proteins are able to form micelles with semicrystalline domains and these micelles further organize into stable supramolecular assemblies via the N- and C-terminal domains.58 As the solution travels through the tapered duct, the micelles elongate and fuse due to phase separation and transition of the dope solution into a solid fiber.58 The increasing shear forces and

postdraw stretching further help align crystalline domains of silk giving the fibers their remarkable properties.59,60 Attempts have been made to copy this process; however, obtaining highly concentrated silk dopes (up to 50% wt/v in silk glands) is not yet possible.61 Being able to fully replicate such a process would greatly improve the properties of synthetic silks to match or even surpass the properties of natural silk fibers. An alternative to the more conventional silks is silk produced by bees, hornets, and ants.62 Unlike the 𝛽-sheet-rich spider and silkworm silks, these silks have four proteins each with a central 𝛼-helical structure that induces the formation of coiled coils. The coiled-coil silks are weaker but more elastic than spider and silkworm silk. For example, hand-drawn fibers from honeybee larvae have a tensile strength of 132 MPa and failure strain of 204% in dry conditions.62 These silks are also readily expressed in E. coli owing to the small protein size (30–45 kDa for each of the four proteins) compared with the full-length major ampullate spidroin proteins (250–320 kDa) that require genetically modified E. coli for expression.56,57,63 Additionally, owing to the sequence similarity among the four proteins it was found that a single protein can also accomplish the same coiled-coil structure eliminating the need to express all four proteins in E. coli for silk fiber formation.64 Although these small and elastic silk proteins are still relatively unexplored in the field of bioengineering, they may be useful alternatives to keep in mind for future applications owing to their proven biocompatibility.65

Resilin and Resilin-Like Polypeptides Resilin is a highly resilient protein that is also a critical component for the extraordinary abilities of insects to fly, move, and produce sound.66 Among the structural proteins described so far, resilin is the newest member to be recombinantly expressed. It was only about a decade ago that resilin from Drosophila melanogaster was expressed using E. coli, a critical milestone that boosted structural studies of this protein and interest in its use for bioengineering applications.66 The protein is composed of three segments with the hydrophilic N-terminal segment being highly elastic, hydrophobic mid-segment containing chitin-binding sequence, and the hydrophilic C-terminal segment that can reversibly undergo conformational changes indicating energy storage66,67 (Figure 5). The phenomenon of energy storage in the C-terminal segment has been explained by thermally induced changes in its conformation from random coil to 𝛽-turns that reverses



Drosophila melanogaster


Resilin nanocomposite

Cross-linkers

Single resilin molecule

FIGURE 5 | Resilin structure from Drosophila melanogaster . A single molecule of resilin (bottom right ) contains three segments: elastic segment (yellow ) from exon 1, chitin-binding segment (gray ) from exon 2, and energy-storing segment (blue) from exon 3. Resilin molecules cross-linked via dityrosine bridges organize into a nanocomposite structures due to self-association of exon 3 protein segment and give insects remarkable abilities of locomotion, flight, and sound production. (Reprinted with permission from Ref 67. Copyright 2012 Nature Publishing Group)

when the protein is cooled. This reversible transition can also be observed when a gel made of the full protein or just the C-terminal segment is mechanically stretched indicating that this mechanical energy may also be absorbed. Qin et al. drew a correlation that similar to the heat absorbed during conformational changes with thermal input, energy from a mechanical input is absorbed and stored in resilin; the stored energy can be released and used for mechanical work upon removal of this input.67 Thus, for insects, resilin is not only a soft highly extensible spacer but also it reversibly stores energy that is used to give a significant boost to the moving appendages enabling insects to jump and/or fly with great efficiency. Recombinantly expressed resilin-like polypeptide (RLP) is composed of tandem repeats of consensus sequences from the N-terminal segment of resilin. These include D. melanogaster-derived ‘GGRPSDSYGAPGGGN’ as well as Anopheles gambiae-derived ‘AQTPSSQYGAP’ repeating units.21 RLPs using both of these sequences have been shown to have similar elastomeric properties. In addition to the elastomeric properties, resilin and RLP are also thermo-responsive. Similar to elastin and ELP, resilin and RLP sequences undergo a sharp transition from hydrophilic to hydrophobic above a transition temperature, a property known as lower critical solution temperature (LCST). However, unlike elastin, resilin and RLP also undergo a similar transition when cooled below a second transition temperature, a phenomenon known as upper critical solution temperature (UCST). Because of the presence of many hydrophilic residues in RLP sequences, the LCST of the protein occurs at a relatively high temperature of 70∘ C, while UCST occurs below 6∘ C.68 One last property of natural resilin is that it undergoes fluorescence resulting from its dityrosine cross-links.

The phenomenon can be replicated in RLP when it is cross-linked via tyrosine residues.66

APPLICATIONS OF PROTEIN-BASED NANOMATERIALS Elastin and Elastin-Like Polypeptides Tissue Engineering rTE has helped reveal many properties of elastin and its derivatives. Along with basic biological studies, rTE has also been used to develop various tissue engineering substrates. The advantages of using rTE are that it does not induce blood clot formation owing to its low thrombogenicity,69,70 it forms compliant structures,71 and it is compatible with many cell types including vascular endothelial and smooth muscle cells.71–73 Additionally, rTE also contains a hydrophilic cell-interactive C-terminal domain with positively charged ‘GRKRK’ domain that induces cell adhesion via integrin binding.23,24,74 The first example of rTE-based scaffolds is electrospun fabrics composed of rTE.71–73 rTE has mainly been spun in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) followed by cross-linking (necessary fiber stability) with amine-reactive 1,6-diisohexanecyanate and glutaraldehyde (GTA),72 and disuccinimidyl suberate.71,73 The disuccinimidyl suberate-crosslinked electrospun fabrics formed using 15% wt/v solutions are soft as revealed by tensile testing of phosphate-buffered saline-soaked fiber mats showing a Young’s modulus of 0.15 MPa, failure strain of 75%, and a tensile strength of 0.38 MPa. Interestingly, the use of higher weight/volume ratios of spinning solution results in ribbon-like instead of cylindrical fiber morphology for rTE also seen in



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electrospun ELPs.75,76 The phenomenon of ribbon formation with rTE and ELP may result from coacervation interactions that are enhanced in elastin solutions with higher concentrations.75 Similar to electrospun ribbons of poly(ether imide), the rTE/ELP may quickly form a thin skin around the spinning jet and the remaining solvent diffuses out of the skin instead of quick evaporation.77 As the elastin skin is soft, the fiber collapses into ribbons as observed with rTE and ELP electrospun fabrics.71,75,77 Another example of rTE-based tissue engineering scaffold is a photo-cross-linkable compliant hydrogel.78 Annabi et al. synthesized methacrylated tropoelastin (MeTro) by reacting rTE with methacrylate anhydride. MeTro can then be mixed with the photoinitiator 2-hydroxy-1-(4-(hydroxyethoxy) phenyl)-2methyl-1-propanone and cross-linked under ultraviolet (UV) to create both 2D cell culture substrates and 3D cell-encapsulating gels.78 Microgrooved compliant MeTro gels that allow for cell alignment have also been used for cardiac tissue engineering with control over cell orientation.79 These gels have variable Young’s modulus from 2 to 14 kPa and can undergo up to 400% strain with low energy loss.78,79 Porous tropoelastin hydrogels have also been produced by chemical cross-linking using disuccinimidyl suberate as described by Tu et al.80 In addition to the direct utilization of rTE, many researchers have also explored its use in improving and tuning properties of materials made of polymers such as polycaprolactone,81 natural polysaccharides such as heparin and dermatan sulfate,82 and proteins such as collagen83 and silk.84–86 Although recombinantly expressed tropoelastin is a homogeneous polypeptide able to mimic natural elastin, only fully synthetic ELPs give the user freedom over sequence design that could be advantageous for developing diverse biomaterials. ELP is also highly biocompatible and therefore has already been used in applications such as cartilage, ocular, and liver tissue engineering.2 A common type of scaffold useful for such applications is a compliant hydrogel, which is achievable for a soft protein such as ELP.31 An important aspect of hydrogel formation using ELP has been the use of lysine for chemical cross-linking. Succinimidyl derivatives such as tris-succinimidyl aminotriacetate (TSAT)87 and N-hydroxysuccinimide functional four-armed poly(ethylene glycol) (PEG)27 have been used for ELP hydrogel formation. The use of organic solvents to avoid hydrolysis of succinimide derivatives has an additional advantage of preventing ELP temperature transition during cross-linking and thus allowing the formation of optically clear gels with constant gel cross-linking.27,87 ELP hydrogels

are also thermo-responsive owing to the ITT property of ELP; coacervation of ELP with increasing temperatures results in gel dehydration and a decrease in gel volume as a consequence.27,87 This reversible property also affects gel mechanical properties as seen with TSAT-cross-linked gels that show a softer range of Young’s modulus of 0.24–3.7 kPa at 7∘ C, while relatively dehydrated gels show a range of 1.6–15 kPa at 37∘ C.87 Many researchers have also sought cell encapsulation using ELP hydrogels to mimic extracellular matrix like 3D environments for tissue engineering.88 For example, lysine-containing ELPs were enzymatically cross-linked using transglutaminase.89 The resulting hydrogels could encapsulate chondrocytes and induce formation of hyaline cartilage-like substrate rich in collagen II.89 Nonenzymatic alternative that also uses amines for cross-linking in mild aqueous conditions is the use of (hydroxymethyl) phosphine derivatives such as [tris(hydroxymethyl)phosphino]propionic acid (THPP)90,91 and tetrakis(hydroxymethyl) phosphonium chloride (THPC).92 The fast reaction between phosphines and amines enables rapid cross-linking that can also be done in situ.91,92 THPP-cross-linked gels with an elastic modulus of about 11 kPa have been used for cartilage engineering,91 while the more recent THPC-cross-linked soft gels with an elastic modulus of about 810 Pa (estimated from shear modulus assuming 𝜈 = 0.5) have been combined with vascular endothelial growth factor (VEGF)-derived QK motif to stimulate human umbilical vein endothelial cell (hUVEC) behavior93 as well as to study neurite growth in 3D environments.94 The THPP-cross-linked gels also show a sequence-dependent elasticity tuning with an increasing distance between cross-linking lysine domains resulting in softer gels.91 In situ cross-linkable ELP hydrogels using cysteine-based disulfide bridge cross-links have also been engineered by Chilkoti and coworkers.95,96 These soft gels with an estimated elastic modulus as high as 450 Pa undergo cross-linking in oxidizing conditions in the presence of H2 O2 with gelation rate and gel stiffness controlled by amounts of H2 O2 and protein concentration, respectively.95,96 The mild concentration of H2 O2 enables cell encapsulation and in situ formation of these soft gels useful in tissue engineering.95 The cell-encapsulating ELP hydrogels mentioned so far use natural amino acids as reactive groups and mild reaction conditions; however, residues such as lysine are an integral part of other biomolecules and the side reactions with cross-linkers can cause unwanted modifications. Ways to avoid this include: using bio-orthogonal chemistry or using physical




cross-linking. Bio-orthogonal chemistry involves the use of reactants that do not interact with biological molecules such as the catalyst-free click reaction between azide and cyclooctyne developed by Bertozzi and coworkers.97 In a recent report, González de Torre et al. functionalized ELP with either azide or cyclooctyne; the copper-free click reaction spontaneously occurs when the two ELPs are mixed to create hydrogels with elastic moduli between 1.8 and 7.5 kPa at 37∘ C.98 The advantage of these gels over the azide-alkyne ‘clickable’ gels developed by Teeuwen et al. is that they do not require the cytotoxic copper catalyst and can safely be used for in situ gel formation.98,99 A major disadvantage for the clickable gels is that ELP functionalization with azides and cyclooctynes requires complex chemical processing compared with other methods and especially compared with physical cross-linking. Physical cross-linking can be accomplished using two elastin-derived plastic, self-associating sequences: ‘VGGVG’100,101 and ‘I/V-PAVG’.102,103 Although not used for gel formation, a triblock elastin-mimic polypeptide with self-associating ‘VGGVG’ end-block sequences and hydrophilic ‘VPG(V/F)G’ mid-block have been shown to self-assemble into beaded fibrils.101 This is expected as ‘VGGVG’ is known to form amyloid-like fiber aggregates.104 Similarly, triblock polypeptides using plastic-like ‘I/V-PAVG’ repeats can form hydrogels as shown in several studies.103,105,106 The gels can assemble thermo-responsively with the plastic ‘IPAVG’ blocks self-associating into physical cross-links.103,105,106 Interestingly, the physically forming gels of elastic/plastic triblock ELPs have also been cast into elastic films. Upon film formation, the film cross-linking can be further enhanced by introducing chemical cross-links between lysines via GTA105 or by introducing hydrazone cross-linking between triblock ELPs functionalized with hydrazide and aldehyde groups.106 The tough physically cross-linked films result in a Young’s modulus (E) of 0.74 MPa and failure strain of 437% and the films increase in stiffness to E = 1.53 MPa and E = 3.16 MPa for chemical cross-linking with hydrazone and GTA, respectively.106 Several other types of films have been synthesized with a focus on the use of ELPs fused with ‘RGD’ and related functional modules. Similar to hydrogels, film cross-linking strategies have relied on lysine residues linking using chemicals such as bis(sulfosuccinimidyl) suberate,107,108 disuccinimidyl suberate,107 and hexamethylene diisocyanate.109,110 ELPs with cell-adhesive motifs such as ‘RGD’ and CS5 domain have also been used as simple surface coatings to study their biochemical effects on cells.111,112

Some recent film/coating work includes ELP membranes for maintenance of retinal pigment epithelial cells,110 fibronectin-mimicking ELP surface coatings to grow and stimulate fibroblasts and neuroblasts,113,114 and in general to improve cell-interactive properties of synthetic polymer scaffold surfaces such as poly(lactic) acid surfaces.115 Improved osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic medium has also been shown on microtextured ELP membranes fused with ‘RGDS’ and ‘REDV’ motifs along with HAP-mineralizing motif.116 A final type of ELP scaffold is electrospun fibrous mats as mentioned previously.75,76,117 It is suggested that the higher surface area of the characteristic ribbon-like fibers of ELP resulting from the use of concentrated spinning solutions may allow for a better display of cell signaling modules to the interacting cells.76

Drug Delivery Elastin-derived peptides have been used extensively as stimuli-responsive polymers useful for therapeutic drug delivery and targeting applications. Similar to its use for tissue engineering scaffolds, rTE has seen little use in targeted delivery applications compared with the more synthetic and customizable sequences of ELP. One of the limited recent examples of rTE involves the use of electrospun rTE for adipose cell delivery to wound sites to promote healing as shown by Machula et al.118 Unlike the limited changes that can be made to rTE, ELP is much more flexible in terms of its sequence and therefore the tuning of its properties. The most powerful quality that makes ELP valuable for drug delivery is its tunable ITT. A model presented by McDaniel et al. is an excellent starting point to design and predict the ITT of a desired ELP based on the hydrophobicity of its guest residues and the molecular weight of the ELP.29 As will be apparent through examples of designs with increasing sophistication, delivery applications using ELP rely heavily on the transition property of this structural PBP for targeting and localization. Temperature-responsive ELP chains have been used directly for cancer targeting and tumor localization using strategies such as localized hyperthermia and fusions with targeting functional groups. For example, Chilkoti and coworkers developed an ELP with careful sequence selection and tuned the transition temperature to about 41∘ C; this allowed the peptide to localize in tumors with the induction of mild hyperthermia.119 The researchers have shown tumor localization and distribution of the thermo-responsive polypeptide using 14 C-labeled ELP and also they show that the in vivo half-life of this ELP is 8.7 h.120 ELP



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accumulation in tumors through the induction of mild hyperthermia without any tags is quite remarkable in itself. A different tumor-targeting strategy is to use ELP functionalized with specific targeting sequences. A recent example of this was shown by Sarangthem et al., who developed ELP with AP1 sequence to target cell surface interleukin-4 (IL-4), an overexpressed cell surface marker in solid tumors.121 By expressing a chimeric ELP with multiple repeats of the targeting sequence AP1, tissue localization is significantly enhanced than using single AP1 sequences.121 In contrast to showing tumor localization via either thermal response or chemical motif, Bidwell et al. have shown a more comprehensive strategy to not only target but also treat glioblastoma.122 The researchers developed an ELP with positively charged cell-penetrating peptide (CPP) at the N-terminus and a therapeutic sequence inhibiting the oncogenic transcription factor c-Myc at the C-terminus.122 The engineered biochemical capability of this ELP to eliminate cancer cells was further enhanced by local induction of hyperthermia at the tumor site to site specifically accumulate the therapeutic ELP.122 The treatment proved its success as it reduced the murine glioblastoma tumors by 80% in volume.122 Free ELP chains are useful for cell targeting and delivering fused therapeutic peptides; however, molecules such as drugs require loadable carriers to deliver them to specific site rather than diffusing to all tissues and affecting normal cells. A successful carrier fabrication method is to use amphiphilic diblock ELP chains with a self-associating hydrophobic block fused with a hydrophilic block. This has been shown in a report using a diblock ELP used to form nanosized micelles loaded with doxorubicin (dox).123 Chilkoti and coworkers designed a hydrophilic ELP domain and a cysteine-rich C-terminal domain ‘[GGC]8 ’ used to conjugate hydrophobic dox molecules. The hydrophobic dox-loaded C-terminus self-associates and the ELP amphiphile forms micelles. The drug-loaded micelles were four times more effective than free drug at eliminating the tumor when injected in a mouse tumor model.123 Similarly, geldanamycin (GA)-loaded ELP particles can also be used for acid-sensitive drug delivery as shown by Chen et al.124 The researchers designed a hydrophilic ELP fused to an aspartate- or glutamate-rich domain and chemically loaded GA on the carboxylic acids of Asp/Glu using acid-labile hydrazone cross-links. The bulky hydrophobic drug-loaded domain gives the polypeptide amphiphilic properties and enables the polypeptides to form micelles.124 The hydrophilic ELP in the drug-loaded micelles was also designed such that it has an ITT of 40∘ C and thus can be

localized by phase transition by inducing hyperthermia at the target sites.124 A different strategy to form ELP amphiphiles instead of drug-induced hydrophobicity as seen with Dox- and GA-conjugated ELP is to design a diblock ELP with the blocks having contrasting ITTs. Such diblock ELPs will have temperature-dependent amphiphilicity as the relatively hydrophobic sequence coacervates at a lower temperature resulting in self-association while the other block remains soluble.125 Amphiphilic ELPs have also been fused with tumor-targeting sequences such as NGR to target CD-13 receptors in tumor vasculature.126 High tumor vasculature retention of these micelles affirms the potential of these nanoparticles for targeted drug delivery in addition to the simpler nonassembling ELP chain-based targeting strategies.121–123,126 A unique and slightly more sophisticated use of ELP nanoparticles was reported recently by Soon et al. using a combination of multiple amphiphilic ELP sequences.127 A ‘core’ block with repeated ‘VPGIG’ was paired with an ‘outer’ block with either ‘VPGAG’ or ‘VPGPG’. The alanine-containing ‘outer’ block was also functionalized with the fibrinogen-binding tetrapeptide, ‘GPRP’. Mixing the two types of diblock ELPs resulted in 55-nm-diameter micelles above the ITT of core blocks while outer blocks stay hydrated.127 Interestingly, because the ELP transition property is impaired for ‘VPGPG’ sequences, only the ‘VPGAG’ sequences undergo temperature transition meaning that the system is able to thermo-responsively present the fibrinogen-binding sequence depending on the hydration state of the alanine-containing outer blocks.127 A strategy like this is powerful when paired with locally induced hyperthermia-based tumor treatments as it would allow not only for thermo-responsive particle localization but also for cell–nanoparticle interaction only at the hyperthermic site and further increase tumor cell specificity of the nanoparticles. Aside from ELP nanoparticles synthesized using diblock polypeptide, there are examples of oil–water emulsion-based particles and ELP capsules useful for drug and gene delivery.128,129 ELP and albumin have been cross-linked using glutaraldehyde to form microparticles using oil/water emulsion for protein delivery. These particles are able to thermo-responsively change their surface pore size and thus control drug release with ELP:albumin ratio-dependent tunability.128 Similar microparticles made of ELP and superparamagnetic iron oxide nanoparticles (enzymatically cross-linked using transglutaminase) have also been generated for future magnetic field-based particle localization strategies and delivery of therapeutic proteins.130 ELP can also




be used to produce capsule shells that can be loaded with therapeutic molecules for delivery.129,131 Using ELP with bioactive motifs can improve the cellular uptake of the capsules as shown by Costa et al. using ‘RGD’-modified ELP.131 Drug delivery using ELP has also been studied in the form of drug depots with stimuli-responsive properties. The ABA triblock sequence mentioned in the last section using plastic ‘I/V-PAVG’ and elastic ‘VPGVG’ is an excellent example. The triblock polypeptides can potentially be used as drug delivery gels and films in addition to their applicability as tissue engineering scaffolds. The work from the Chaikof group that has resided on incorporating physical and chemical cross-linking105,106 into the ELP gels and films has led to a recent report studying the effects of such cross-linking on the release rates of drug in addition to mechanical properties of the films.132 Similarly, cysteine-containing ELPs that spontaneously form hydrogels in situ with disulfide bridge formation can also be useful for drug delivery purposes.95 The rate of gel formation can be controlled by H2 O2 concentration, which turns out to be in moderate concentrations (80% of the loaded drug (ethacridine lactate) was released in 1 day at pH 2, whereas only 10% was released in the same time at pH 7 and above.181 The drug-loaded nanoparticles can also be enzymatic degraded by trypsin and elastase making the particles useful for cell activity-mediated drug release.181 Hofer et al. used similar particles composed of eADF4(C16) for lysozyme delivery.182 The positively charged lysozyme was easily loaded in the eADF4(C16) nanoparticles and the in vitro release tests showed a pH- and ionic strength-dependent protein release.182 The researchers also showed that the SLP nanoparticles can be lyophilized for long-term storage without any damage.182 On the other hand, N. clavipes MaSp1 protein-derived sequence has been used to design self-assembling nanoparticles useful for gene delivery.183,184 Numata et al. synthesized a triblock SLP containing six repeats of the MaSp1 consensus, poly(l-lysine) with 30 lysine residues, and either a tumor lymphatics-targeting domain or cell death-inducing domain. The triblock polypeptides self-assemble to form nanoparticles in the presence of negatively charged genes.184 Particles with tumor lymphatics-homing peptide showed better transfection efficiency resulting in their use in a mouse model for in vivo gene delivery.185 Additionally, the particles also have a tunable enzymatic degradation rate dependent on its 𝛽-sheet content that can be regulated by methanol treatment of the particles.186 Other scaffold structures made of SLP being developed for drug delivery are films. Similar to the SLP nanoparticles for gene delivery, films can also have tunable degradation and drug release by changing the film crystallinity.186 A standard method used to increase 𝛽-sheet content of silk structures is via dehydration using nonsolvents such as methanol.187 Scheibel and coworkers have studied the effects of methanol and different film casting solvents such as HFIP, formic acid, and water on the mechanical properties of eADF4(C16) films.188 And although not tested by Spiess et al., the effects observed on the film mechanical properties due to changes in SLP crystallinity and packing would simultaneously affect release rates of loaded drugs. Another way of tuning release rate of drugs from SLP films is to mix the polypeptide with a synthetic polymer as shown by Hardy et al.189 The films cast with a combination of eADF4(C16) and polycaprolactone or pellethane show degradation and drug release dependent on the amount of synthetic polymer; lower polymer

results in faster degradation and drug release and vice versa.189

Resilin and Resilin-Like Polypeptides Tissue Engineering RLPs are relatively unexplored in the field of biomaterials. Researchers who use RLP have focused on developing hydrogels for tissue engineering. As described earlier, RLP gene from D. melanogaster resilin has three exons, each with a different purpose.67 Qin et al. synthesized full-length resilin from D. melanogaster resilin genes to synthesize hydrogels.190,191 The expressed resilin was cross-linked by forming dityrosine bridges in two ways: enzymatically, using horseradish peroxidase, and photo-Fenton reaction, using FeSO4 and H2 O2 followed by UV exposure.191 The researchers showed that among the three exons, hydrogels from exon 1 showed 90% resilience, whereas those from exon 3 showed only 60% resilience.190,191 These studies paved the way to the mechanism of resilin function proposed by Qin et al.67 Studies such as those by Kiick and coworkers have further revealed the excellent properties of the elastomeric ‘GGRPSDSYGAPGGGN’ sequence derived from exon 1 of D. melanogaster resilin. The RLP12 containing 12 consensus sequence repeats has been cross-linked in mild conditions using [tris(hydroxymethyl)phosphino]propionic acid192 or tris(hydroxymethyl)phosphine7 cross-linkers in coordination with primary amines on the RLP12. Charati et al. engineered RLP12 to contain cell-binding motifs, MMP degradation sites, and heparin-binding sites spread throughout the polypeptide.192 These gels have a storage modulus of about 10 kPa and were observed to withstand up to 450% strain. On the other hand, Li et al. developed tunable hydrogels using four different RLPs: one each with a cell-binding site, a MMP site, a heparin-binding site, or nonfunctional RLP.7 These hydrogels were shown to have an RLP- and cross-linker-density-dependent storage moduli in the range of 1–25 kPa.7,193 Because of their intrinsic high resilience and a simple ratio-dependent tuning of functional motif content, these hydrogels were indicated for use in vocal fold engineering and many other tissue engineering applications.7 Slightly modified RLP hydrogels for vocal fold tissue engineering with RLP containing lysine- and glycine-rich cross-linking motifs have been further shown to have a resilience of up to 98% by Li et al.194 These gels show better energy storage and recovery than synthetic polymer gel controls and their tensile properties were comparable to vocal fold tissue.194 An additional type of RLP hydrogel produced by McGann et al. was made using cysteine (for sulfhydryl-based cross-linking)-containing




RLPs of different lengths: 12, 24, 36, and 48 monomer repeats.195 Interestingly, the gels were measured to have the similar storage moduli (7–9 kPa) using 1:1 RLP:cross-linker ratio (cross-linker: vinyl sulfone functional four-armed PEG) because the RLP molecular weight between each pair of cysteine residues was kept the same. The mild cross-linking chemistry allows for cell encapsulation and the range of measured storage moduli makes the gels ideal for cardiovascular tissue engineering.195 Another type of RLP with the consensus sequence from A. gambiae resilin, ‘AQTPSSQYGAP’, has also been used to create hydrogels. Renner et al. synthesized RZ10, an RLP with 10 repeats of the A. gambiae resilin-derived sequence. The RZ10 hydrogels were shown to have a dynamic modulus of 22 kPa and surfaces coated with RZ10 fused with RGD showed proper cell spreading.196 Additionally, similar to the development of bioactive ELP, control of stem cell differentiation such as hMSCs into osteoblasts has also been shown on surfaces coated with RZ10 fused with bioactive motif from bone morphogenic protein-2.197

MODULAR DESIGN OF PBPs Modular PBP design using genetic engineering allows for nanoscale control of protein functionalities. Addition of cell-adhesive peptides such as ‘RGD’, ‘REDV’, and ‘IKVAV’ has become common for biomaterial functionalization as can be seen among the examples throughout the previous sections. Some of the more interesting research involves modular PBPs composed of a combination of structural modules or a structural module with functional modules that can undergo biomineralization or form organic–inorganic composites. Many such nature-inspired and unnatural functional modules alike have been developed using display techniques such as phage display. Phage display is an evolutionary high-throughput screening process to identify functional peptide sequences. Through the insertion of random DNA sequences into the phage genome, we can express random peptides on the surface of phage coat proteins and identify specific peptide sequences.198,199 An example of phage-display application comes from the work of Jaworski et al. to find the trinitrotoluene (TNT) explosive-binding peptide ‘WHWQ’ sequence.200 Oh et al. used the ‘WHWQ’ functional phages to create optically responsive bio-inspired nanostructured patterns, which can be useful for colorimetric sensors. The colorimetric patterns thus fabricated were shown to sense TNT in quantities as low as 300 ppb.49,201 Similar functional modules found using phage display include biotin-like peptide ‘HPQ’,202,203

breast cancer tumor-localizing peptide ‘CREKA’,204 cyclic RGD peptide ‘CDCRGDCFC’ with better integrin binding than linear RGD,205 and carbon nanotubes/graphene-binding ‘HNWYHWWPH’ peptide,27,206,207 to name a few. Using these as well as creating new functional modules, we can take full advantage of modular peptide design to create new PBPs with nanoscale and subnanoscale precision and materials with properties that were out of reach until now. In this section, we will describe various modular PBPs that have been developed with focus on PBPs containing multiple structural domains among ELP, SLP, CLP, and RLP along with a few examples of organic–inorganic composites. Recently, the modular synthesis of the PBPs and their applications as biomedical materials was reviewed by DiMarco and Heilshorn.3

Tissue Engineering Much research has gone into the development and application of new types of modular polypeptides that combine the properties of silk, elastin, collagen, and/or resilin. A highly used chimeric polypeptide is the silk–elastin-like polypeptide (SELP).208 It has already been more than two decades since the development of SELPs and numerous structures and uses have been shown. The silk block (S) is generally composed of the silkworm silk-derived ‘GAGAGS’, while the elastin block (E) is composed of ‘VPGVG’ with certain ELP pentapeptides containing guest residues like lysine that can be used for chemical cross-linking. The general formula for the peptide can be stated as [Sm ,En ]o , where ‘m’ and ‘n’ define the number of monomer repeats of S and E, respectively.209 The SE monomers can then be repeated ‘o’ number of times to obtain SELPs of various lengths/molecular weights. Numerous possible combinations make it difficult to choose a sequence and Wang et al. have recently tried to address this by developing a high-throughput method to screen the different types of sequences.210 Wu and coworkers have used SELP-47K, a 69.8-kDa polypeptide with 4:8 ratio of silk:elastin blocks, to produce films useful as tissue engineering scaffolds.211 The SELP films of tunable thicknesses can be produced by simply drying a solution of the polypeptides. An additional step required to produce stable films is film treatment in methanol for proper physical cross-linking.211 Methanol causes an increase in aggregated 𝛽-strands and a decrease in 𝛽-sheet content as identified via FTIR and the films thus produced were shown to be stable with water content of about 51%.212 The authors showed that the physically



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cross-linked films have an elastic modulus of 1.7 MPa with failure strain of 190% and resilience of 86%.211 Chemical cross-linking with GTA performed after methanol treatment results in stiffer films with lower water content (38%).212 The films were shown to have little toxic effects toward NIH 3T3 fibroblasts, which supports the applicability of these as tissue engineering scaffolds.212 Other ways that SELP has been processed include wet spinning and electrospinning to generate fibrous scaffolds. Similar to film synthesis, SELP fibers need to be treated with a nonsolvent like methanol to induce proper physical bonding. SELP-47K has been wet-spun in methanol coagulation bath to obtain strong and extensible fibers with tensile strength of 20 MPa and failure strain of 700%.213 On the other hand, electrospinning of SELP has been accomplished using SELP with silk:elastin block ratios of approximately 1:2 dissolved either in formic acid or in water.214,215 For each type of spinning solvent, the fibrous mats were treated with methanol and/or GTA vapors. The mats show elastic modulus in the range of 3.4–13.2 MPa and failure strains of 100–130% as well as high biocompatibility for in vitro cell culture.214,215 SELPs have also been processed into physical hydrogels; although many thermo-triggered gels have already been developed SELPs, the process of gel formation was not studied more carefully until recently. Xia et al. studied the effects of silk:elastin ratio on the ability of the SELP to self-assemble into higher ordered structures. It was theorized that SELP thermo-responsively self-assemble into 100–200 nm diameter micelles that assemble further to make nanoparticles, nanofibers, and hydrogels.8 The study reveals the importance of sequence selection and temperature-based structure formation to determine the reversibility of the structures; for example, hydrogels tend to be irreversible, but the nanoparticles can be resolubilized.8 The same observed phenomenon may be involved with the thermally triggered irreversible physical hydrogels reported previously for chondrogenesis and biomolecule delivery systems.216–218 Some of the other modular PBPs include chimeras of silk and collagen and a polypeptide composed of domains from resilin, elastin, and collagen. Just like the more extensively used SELPs, these PBPs also exhibit a combination of properties from each of the individual components. For example, the silk–collagen-like chimeric polypeptides developed by An et al. have a silkworm-derived ‘GAGAGS’ silk block with bacterial collagen-derived collagen-mimetic block.9 These PBPs can bind to silk fibroin scaffolds by self-association between the

silk domains, while the collagen-like cell-interactive properties of the triple helical CLP block impart bioactivity to the material.9 These polypeptides can easily provide collagen-like cell-interactive properties to any silk fibroin material through simple physical interactions. A different type of silk–collagen-like polypeptide was developed by Martens et al. using a modified silkworm-derived silk block, ‘GAGAGAGE’ that is pH-sensitive, and a collagen-derived sequence rich in ‘N’, ‘Q’, and ‘S’ residues has been used to form nanotapes and physical hydrogels.219,220 The collagen-derived sequences in this case remain in an unordered hydrated state like gelatin, whereas the silk domains form physical cross-links through 𝛽-sheet stacks of the silk-like domains.220 The gels were able to achieve high stiffness with a storage modulus around 40 kPa just from physical cross-links.220 Similar gels with storage moduli below 10 kPa have been characterized further to show self-healing properties as the physical cross-links between silk domains can reform; however, the noncytotoxic gels have little intrinsic bioactivity as shown in a cell study using rat primary mesenchymal stem cells.221–223 Unlike the silk–collagen-like chimeric polypeptides with rigid, self-assembling silk and bioactive9 or random coil220 collagen-like domains, the more recently established resilin–elastin–collagen-like polypeptides (RECs) are composed of flexible resilin and elastin domains along with rigid, self-assembling collagen-like domains.10 As expected, the individual REC chains have shown exceptional extensibility along with the self-assembling properties resulting from collagen-like domains.10,224 RECs self-assemble into fibrous aggregates that support cell proliferation as shown with primary human MSCs.224 The cell alignment is also shown to be affected by the orientation of the underlying fibers giving the materials a potential to physically influence cell behavior and increases their value as tissue engineering scaffolds. In addition to chimeras of structural protein modules, there are many examples of PBPs that are chimeras of a structural module with a functional module to enable organic–inorganic composite material synthesis. Recombinant collagen type I has already been used to study and direct HAP nanocrystal formation to determine mechanisms of bone formation.225,226 Similarly, Huang et al. synthesized a chimeric SLP composed of 15 repeating units from N. clavipes MaSp1 protein fused with C-terminal domain of dentin matrix protein 1 (CDMP1) as a functional module able to mineralize HAP.167 The film made of the SLP-CDMP1 chimera was able to form HAP, fluoroapatite, and carbonate–apatite




when incubated for 21 days in 1.5× simulated body fluid.167 N. clavipes-derived SLP (with six repeats) can also be combined with bone sialoprotein consensus sequence and the resulting films form CaP minerals within 6 h of incubation in accelerated calcification solution.227 hMSCs cultured on the mineralized films show osteogenic differentiation over 14 days and subcutaneously implanted nonmineralized films show no signs of inflammation in mice making the films fit for bone regeneration applications.227,228 CaP mineralization has also been carried out using ELP-based chimera containing ELP with N-terminal 15 amino acid domain from salivary statherin. The gel-like coacervates of the ELP chimeras were efficient at CaP mineralization compared with soluble protein.229 Wang et al. developed an organic–inorganic composite bone cement using ELP and HAP.6 The genetically engineered fusion PBP of ELP- and HAP-binding octaglutamic acid was able to physically coat HAP and help it remain dispersed in solution at ambient temperature and transition to form a stable cement at the physiological temperature of 37∘ C. The ELP–HAP bone cement was shown to be injectable with high washout resistance owing to cohesion between ELP coacervate around HAP and to have a compressive strength of 57 MPa, about four times greater than cancellous bone.6 Recently, Wang et al. also synthesized a new chimeric ELP fused with graphene binding (GB) functional module ‘HNWYHWWPH’. This ELP–GB can physically bind to reduced graphene oxide (rGO) and graphene oxide (GO) via 𝜋–𝜋 stacking and keep the nanomaterials dispersed.207 The composite created as a result is pseudo-light-responsive as rGO and GO act as photothermal heaters by absorbing light and locally generating heat, while ELP absorbs the heat and responds mechanically by contracting. The ELP–GB chimera was further modified with the addition of ‘RGDS’ cell-adhesive module to show the ease of improving cell behavior on these composites.207 The ELP–GB/rGO composites can also be used to create light-responsive hydrogels along with an additional ELP containing a C-terminal lysine for cross-linking. As shown by Wang et al., the primary amine groups were cross-linked together using an N-hydroxysuccinimide functional four-armed PEG linker in organic solvents.27 A simple method of creating anisotropic gels was employed by exposing the cross-linking mixture of ELP, rGO, and PEG to humidity. As water vapor diffuses into the cross-linking mixture, ELP transitions resulting in a gel with a distinct porous layer and a solid bottom layer unaffected by water.27 This gel is near infrared

light responsive as rGO absorbs 808-nm light and generates heat that causes ELP to transition and the gel to contract. However, instead of an isotropic contraction, the porous layer of the gel contracts much faster than the solid side resulting in bending of the gels. Wang et al. also demonstrated precise spatial and temporal control of the light-responsive actuation of these hydrogels27 (Figure 6), raising their potential as remotely controllable soft actuators for cell biophysics studies to understand cell behavior in mechanically active tissue-like environments.

Drug Delivery Drug delivery applications that use modular PBPs have mainly focused on aforementioned silk/elastin structural chimeras, SELPs. Tough and crystalline properties of silk along with stimuli-responsiveness and flexibility of elastin combine to create a material that has great potential for the field. Similar to ELP- and SLP-based structures, SELP has also been used to produce nanoparticle constructs as drug delivery vehicles. For example, Anumolu et al. synthesized SELP nanoshells with narrow dispersity using electrospray droplet evaporation technique combined with a differential mobility analyzer.230 The differential mobility analyzer separates particles by classifying them electrostatically allowing collection of narrowly distributed particles. Anumolu et al. were able to obtain particles with diameter between 20 and 40 nm and suggest the potential of these particles as carriers for bioactive and imaging agents.230 A recent report by Xia et al. (who previously developed SELPs with different silk:elastin block ratios8 ) shows how their SELPs can be used to generate drug-loaded micelles. The researchers were able to show that a hydrophobic drug such as doxorubicin can induce micelle formation without the need for thermal stimulation, making the nanoparticle assembly process simple.231 The drug-loaded particles show 1.8 times higher cytotoxicity in HeLa cells compared with free drug.231 SELPs have also been processed into films that have potential in controlled drug delivery. As mentioned in the previous section, SELP-47K used by Wu and coworkers was used to produce robust biocompatible films.211 In addition to being mechanically stable, the films were also shown to be optically transparent. The film transparency was unaffected by film-stabilizing post-treatments such as with methanol to induce physical bonding as well as with GTA to induce chemical bonding.212 Recently, these films were applied as optically clear membranes for the delivery of ocular antibiotic, ciprofloxacin.



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Tuning of drug release from the films was performed by film post-treatment in either MeOH or EtOH.232 These films are valuable owing to their ability to sustain cell growth and elute drugs.212,232 One last SELP structure used for drug delivery is a hydrogel. Ghandehari and coworkers have used SELP with different ratio of silk:elastin blocks to form hydrogels for delivery of adenoviruses for gene therapy. The role of the SELP was to form depots in situ that slowly release the therapeutic adenoviruses locally for effective treatment of diseases such as cancer.217,218 The ratio of silk and elastin blocks affects gel properties such as gelation time, stiffness, pore size, hydration, and even biodegradability.217 More recently, a new polypeptide was generated by fusing SELP with matrix metalloproteinase (MMP) sites to enable MMP-based biodegradation of the material. The resulting hydrogels are sensitive to MMP-2 and MMP-9 and can thus allow for enzymatic degradation-based drug release from the gel depots.233 Similarly, the light-responsive ELP/rGO hydrogels can also be used as drug delivery depots.27 The highly localized response of the gels to 808-nm laser light can allow for drug release from specific sections of the gels. Additionally, the use of near-infrared light

that can penetrate skin would allow the drug-loaded gel to be implanted subcutaneously and activated noninvasively as required.

CONCLUSIONS AND FUTURE PERSPECTIVES In this review, we discussed some of the well-characterized PBPs and their representative biomedical applications. Proteins are fundamental parts of all biological systems and structural proteins such as collagen and elastin play important roles in maintaining the integrity of our body. Moreover, even insect-derived proteins such as silk and resilin have proved to be extremely useful owing to their intrinsic biocompatibility. Understanding the properties of these proteins and being able to design new mimics can help us tackle the challenge of replicating ECM-like environments as well as creating completely novel smart materials through protein self-assembly and stimuli response. The functional biosignal domains in addition to the smart structural domains add to the value of PBP allowing one to stimulate cells physically and biochemically.

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FIGURE 6 | Example of the modularly synthesized protein-based polypeptide (PBP) and graphene nanocomposite to show the light-controlled motion of hydrogels. Images of the fingers of a hand-shaped hydrogel, made of PBP-graphene, bending and unbending in response to near-infrared light stimulation. (Reprinted with permission from Ref 27. Copyright 2013 American Chemical Society)




Modular design of PBP using motifs from different structural and functional motifs is paving the way for the future of precisely and purposefully designed bionanomaterials useful for tissue engineering as well as biomolecule delivery. A drawback of this technology keeping it out of reach from most researchers is the relative difficulty of PBP synthesis compared with off-the-shelf polymers. Nevertheless, a significant amount of research has been undertaken toward efficient recombinant expression of natural protein mimics such as ELP, CLP, SLP, and RLP.1,52 In the future, this will certainly

help make PBPs available for a wider audience in academia and industry alike instead of the current limited use by genetic engineering/protein biomaterial experts. Additionally, the fast-growing field of computation-aided structural protein design will significantly enhance efforts in protein-based nanomaterial design by automating structure and function prediction of desired PBPs.234,235 We will surely witness new innovative material science solutions and advanced applications of these technologies in the near future.

ACKNOWLEDGMENTS This work was supported by National Science Foundation Center of Integrated Nanomechanical Systems (EEC-0832819), and NIH ARRA supplement to an NIDCR R21 grant (DE 018360-02).

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