Engineered Protein Biomaterials - Tissue Engineering Principles and Practices

Andreina Parisi-Amon

Stanford University

Sarah C.

Heilshorn

Stanford University

4.1 Engineered Protein Biomaterials as an Alternative to

“Traditional” Biomaterials ... 4-1 4.2 Synthesis of Engineered Protein Biomaterials ...4-3 4.3 Design of Engineered Protein Biomaterials ...4-5

Crosslinking Domains • Structural Domains • Degradation Domains • ECM Cell-Binding Domains • Cell−Cell Adhesion Domains • Cell-Directive Domains

4.4 Applications of Engineered Protein Biomaterials ... 4-11 References ... 4-11

while the complex biochemical compositions of these materials are valued for their ability to initiate multiple cellular signaling pathways, their compositions also make naturally derived materials nonideal, as their properties cannot be easily tailored. Moreover, harvesting and processing of these materials may destroy their higher-order structures (such as fibers) while also producing undesirable batch-to-batch variability. In addition, some natural materials are known to cause high levels of immunogenic response and can have additional clinical translation difficulties due to their mammalian origins. Borrowing bio-functional peptide domains from natural ECM proteins and including them within engineered proteins enables the creation of multifunctional biomaterials that address many of these concerns.

Crosslinking Domains

Liu et al. 2003

Straley and Heilshorn 2009

Charati et al. 2009

Petka et al. 1998

Wong Po Foo et al. 2009

Ehrbar et al. 2005

X 2

• Calmodulin-binding domains

• Leucine zippers

• WW + Proline-rich binding pairs

• Transglutaminase targets

Structural Domains

Degradation Domains

ECM Cell-Binding Domains

Cell–Cell Adhesion Domains

Cell-Directive Domains

• Random coils

• α-helices

• β-sheets

• Silk-like domains

• Elastin-like domains

• Resilin-like domains

• Enzymatic targets (e.g., tPA, uPA, MMP)

• RGD

• PHSRN

• CS5

• IKVAV

• YIGSR

• Cadherins (e.g., E-cadherin)

• Cell adhesion molecules (e.g., NCAM)

• Hormones

• Signaling molecules (e.g., hJagged1, hDelta1)

• Growth factors

(e.g., FGF, VEGF, BMP2)

FIGURE 4.1 In the modular protein engineering design strategy, multiple peptide domains are fused together to design novel, multifunctional, recombinant protein polymers with specific properties.

4-3 Engineered Protein Biomaterials

An alternative approach to create tailorable biomaterials is the use of synthetic polymers such as PEG (polyethlyene glycol), PMMA (poly(methyl methacrylate)), PHEMA (poly(2-hydroxyethyl methacry-late)), and PLGA (poly(lactic-co-glycolic acid)) derivatives. While these materials are easily tailored, they are usually bio-inert without further modification. As such, these materials can only achieve biofunc-tionality with the incorporation of additional components such as ECM-derived peptides and proteins.

Often however, these functional peptides play a role in the mechanical structure, making it difficult to independently tune the biofunctionality and mechanical properties of the biomaterial (Thompson et al. 2006). In addition, the synthetic chemistries inherent to these materials may carry the risk of toxic crosslinkers, activating agents, and degradation fragments (Williams et al. 2005, Seymour et al. 1987).

While protein-engineered biomaterials overcome some of the concerns associated with natural and synthetic biomaterials, they also have their own limitations. Before these materials can be considered for clinical translation, the laboratory-scale synthesis and purification processes typically used during the biomaterial design phase must be optimized to achieve efficient scale-up of production. Although the materials are generally made from protein building blocks native to the human body, rendering them cytocompatible and bioresorbable, they may nonetheless trigger an immunogenic response, particularly due to their synthesis in a foreign host organism. For example, proteins made in Gram-negative bacte-ria, such as Escherichia coli (E. coli), must be sufficiently purified to remove endotoxin, a lipopolysac-charide that can trigger an innate immune response (Rietschel et al. 1994). Even with these challenges, protein-engineered materials constitute an exciting area of biomaterials research given their exquisite design control that enables the creation of novel biomimetic cell scaffolds. In this chapter, we will focus on recent developments in the field of engineered protein biomaterials and highlight opportunities for future advances.

4.2 Synthesis of Engineered Protein Biomaterials

Following the design of a specific protein polymer (which is discussed in the following section), a vari-ety of methods can be used to synthesize and purify the protein. Solid-phase synthesis is the process by which novel proteins are manually created through the sequential addition of individual amino acids (Kates and Albericio 2000). While this process has become more optimized and commonplace over the past several years, the resulting proteins are limited in length and the process is too time consuming and expensive to scale-up to the high levels of production needed for potential therapies. Instead, with the discovery of molecular cloning in the 1970s, scientists have been able to harness the protein factories that exist in nature—cells (Porro et al. 2005). Mammalian (Nagaoka et al. 2002), insect (Tomita et al.

1999), plant (Karg and Kallio 2009), fungal (yeast) (Graf et al. 2009), and bacterial (Zerbs et al. 2009) cells have all been used for recombinant protein synthesis, each with their advantages and disadvan-tages. Irrespective of the host, the creation of a protein through the cellular processes of transcription and translation is inherently advantageous, as it provides efficient molecular-level control of the protein synthesis. Furthermore, built-in accuracy and error-checking mechanisms by the ribosome ensure that the desired protein sequence is being produced (Zaher and Green 2009).

Choosing which host to use is a key step in recombinant protein synthesis, as it determines the com-plexity of the protein sequence that can be produced, as well as the efficiency with which the production can take place. Microorganisms such as E. coli and Saccharomyces cerevisiae (yeast), with their relative ease of genetic modification, low cost of culture, and high growth rates compared to mammalian cells, are often chosen as the host. In fact, for simple protein structures, prokaryotic E. coli is often the first host of choice due to its simplicity and versatility. However, for more complex proteins that require post-transcriptional possessing for correct structure and resulting function, eukaryotic yeast, such as S. cere-visiae and Pichia pastoris are more often chosen, as they combine the high growth rate and simplicity of a single-celled microorganism with the organelles needed for specialized folding and modification.

Once the host is chosen, the exact nucleotide sequence must be designed, while keeping in mind that various hosts may have different tolerances to specific sequences. While some basic tenets are known,

such as the fact that highly repetitive sequences have an increased susceptibility of resulting in unwanted recombination events (Bzymek and Lovett 2001), it is difficult to predict a priori which sequences will have high translational efficiency and yield, therefore making sequence design an iterative process. To that end, scientists are working to create sequence design programs that use host-specific algorithms to improve expression (Gao et al. 2004). Once designed, the completed sequence is synthesized and introduced into the host cell for production. Culture conditions, such as pH, temperature, and oxygen abundance also play a complex role in the yield of protein production.

After protein production, the product must be collected from the cell, either through secretion or cell lysis, and then purified such that only the protein of interest remains. Purification can be achieved through various chromatographic methods, in which the product-containing solution is run through a resin-packed column that takes advantage of specific properties of the target protein, such as size, charge, hydrophobicity, or ligand binding. The basic process includes binding or capturing the protein of interest to the resin, allowing all impurities to run through, and then releasing the purified product for collection (Nilsson et al. 1997). Often multiple iterations are required to isolate the target protein with the desired level of purity. To scale up the process for larger yields, chromatographic methods are often deemed too expensive and time-intensive; therefore, alternative techniques utilizing differential target protein solubility are often developed. For example, target proteins that include an elastin-like sequence typically exhibit lower critical solution temperature behavior, whereby the protein forms a highly con-centrated coacervate at elevated temperatures while most other contaminating proteins remain in solu-tion (McPherson et al. 1996). This thermodynamic phenomenon can be exploited to purify the target protein through a simple sequence of centrifugations at alternating temperatures above and below the target protein’s lower critical solution temperature (Meyer and Chilkoti 1999). Finally, additional purifi-cation may be needed to make the product cytocompatible for proteins expressed in Gram-negative bac-teria such as E. coli. These target proteins are often contaminated with residual amounts of endotoxin (i.e., lipopolysaccharide), a component of the bacterial cell wall that can activate an innate immune

Design and optimization

FIGURE 4.2 Design and synthesis of recombinantly engineered protein polymers. First, an expression host and target amino acid sequence are chosen. This information is used to design a DNA template that encodes the engi-neered protein polymer. After synthesis and cloning of the DNA template into a recombinant expression plasmid, the plasmid is introduced into the host organism. A fermentor is used to control environmental parameters during host proliferation and protein expression. Following protein extraction and purification, a pure sample of engi-neered protein polymer remains.

4-5 Engineered Protein Biomaterials

response. Several techniques have been developed for efficient endotoxin purification, with the most commonly used being an affinity-based column (Petsch and Anspach 2000).

Through iterative optimization, the use of microorganism hosts for recombinant protein engineering provides an economical and efficient method to synthesize engineered proteins in therapeutic quantities (Figure 4.2). Optimized protocols and laboratory-scale fermentors enable the growth of high-density cultures in volumes from 1 to 200 L, enabling the production of multigram protein yields (Heilshorn et al. 2003, Chow et al. 2006, Welsh and Tirrell 2000, Shiloach and Fass 2005).

4.3 Design of Engineered Protein Biomaterials

The inherent modularity of the peptide building-block design strategy of protein-engineered biomateri-als provides the ability not only to design materibiomateri-als emulating a specific biological niche, but biomateri-also to cre-ate a versatile family of mcre-aterials simply through the inclusion or removal of singular peptide domains.

The domains that are fused together to create full-length proteins can be classified by the functionalities they convey to the final product (Figure 4.1). For example, many biologically inspired domains can interact directly with cells through the promotion of cell−ligand interactions, cell−cell adhesion mim-icry, or behavioral instruction (i.e., regulation of proliferation, differentiation, etc.). Alternatively, other domains can affect material properties, such as degradability and elastic modulus (i.e., the stiffness of a material), which may further direct cell behavior (Discher et al. 2005). Other selected domains can impart specific structural motifs, such as random coils (Davis et al. 2009), coiled-coils (Stevens et al.

2004), β-sheets (Marini et al. 2002), and hierarchical self-assembling domains (Chung et al. 2010) to the protein polymers, which affect the material’s microstructure.

Historically, these peptides were identified by isolating domains of interest from naturally evolved pro-teins. The tripeptide RGD sequence (arginine−glycine−aspartic acid), a commonly used cell- adhesion domain, is a prime example of this. RGD was isolated in 1983 from the extracellular and plasma protein fibronectin and was identified as the minimal sequence necessary to promote cell-attachment proper-ties (Pierschbacher and Ruoslahti 1984). Other commonly used domains include elastin-like sequences, which are derived from the protein elastin found in connective tissue (Meyer and Chilkoti 2002), and recombinant-silks (Prince et al. 1995). Both of these peptide domains are used to confer their unique mechanical properties (i.e., resilience, elasticity, and strength) to the resulting biomaterial.

More recently, the design of protein-engineered biomaterials has not been limited to domains found in nature. As computational design (Hin Yan Tong et al. 2002) and high-throughput screens (Sidhu et al.

2003) are increasingly being used in peptide development, the variety and specificity of domains avail-able for biomaterials design are rapidly expanding. The design process, however, is not always straight-forward. For example, the functionality of a given peptide can be affected by the context of the fully assembled protein, that is, the identity of the flanking peptide domains (Heilshorn et al. 2005). As such, the activity of the domains in each protein composition must be evaluated after the initial design phase.

Another complication in peptide selection is the lack of clarity surrounding exactly which properties are imperative for specific niches. Because protein-engineered biomaterials are synthesized to include ratio-nally chosen domains, this design strategy enables iterative testing and optimization of cell− material interactions to overcome both of the limitations discussed above. To illustrate this inherent design flex-ibility, the sections below give several specific examples of peptide domains identified from naturally evolved proteins, through computational design, or by high-throughput screening to confer specific biomaterial functionalities.

4.3.1 Crosslinking Domains

The inclusion of crosslinking domains enables the formation of a network from the individual designed protein polymer chains, forming two- and three-dimensional material structures with the desired mechanical integrity for supporting cells. Because many cellular behaviors, including spreading,

signaling, and gene transcription, are known to be responsive to the stiffness of the biomaterial, it is critical to exert control over this design variable in order to direct cell growth and differentiation (Discher et al. 2005). The monodispersity of recombinant proteins, resulting in polymers with identical composition, allows for the tight regulation of the frequency and distance between crosslinking sites, with higher crosslinking densities generating stiffer materials (Welsh and Tirrell 2000). Several cross-linking strategies exist for protein-engineered biomaterials, including enzymatic covalent crosscross-linking, chemical covalent crosslinking, and physical (i.e., noncovalent) crosslinking via peptide domains that associate through electrostatic or hydrophobic/hydrophilic interactions.

An example of enzymatic covalent crosslinking is the use of the enzyme transglutaminase (TGase).

TGase is found naturally in the processes of wound healing and ECM stabilization, where it catalyzes covalent bond formation between lysine (K) and glutamine (Q) residues through a calcium-dependent reaction (Greenberg et al. 1991). Through a process of rational peptide design and screening, several amino acid sequences were identified to have high specificity and tight binding to TGase (Hu and Messersmith 2003). In one example, these optimized TGase crosslinking peptides were included as domains within a family of engineered proteins with varying molecular weights between the lysine-containing domains, resulting in a family of biomaterials with a fourfold range in modulus, from 4 to 16 kPa (Davis et al. 2010).

The binding of calmodulin protein to calmodulin-binding domains (CBDs) is another calcium-dependent crosslinking reaction, although this strategy results in physical (rather than covalent) cross-links. Upon binding four calcium ions, calmodulin undergoes a conformational change, allowing it to bind to the hundreds CBDs found in other proteins. This binding is reversible upon the depletion of calcium ions. The myriad of both natural and engineered CBDs improves the versatility of this binding method, as calmodulin−CBD pairs can be chosen with binding affinities that range over five-orders of magnitude and with differing calcium dependencies, ultimately enabling control over the material’s modulus and the reversibility of network formation (Topp et al. 2006).

Leucine zippers comprise another interesting crosslinking domain that allows for reversible self-assembly, in this case through the noncovalent association of coiled-coil domains (Petka et al. 1998).

Naturally evolved leucine zippers function as DNA-binding domains in various transcriptional regula-tory proteins. The motif has been well characterized and is known for its heptad amino acid repeat with hydrophobic amino acids at positions one and four and charged amino acids at positions five and seven (Landschulz et al. 1988). At specific pH and temperature conditions, the zipper peptide folds into a helical structure with both hydrophobic residues on one face, promoting interhelical interactions between mul-tiple folded zippers and leading to association. Connecting concatenated zipper motifs by a hydrophilic amino acid sequence creates a triblock co-polymer that utilizes the natural protein−protein interactions for the formation of a hydrogel, where the zipper domains provide the physical crosslinks (Petka et al.

1998). This system lends itself to independent tuning of both the hydrophilic domain (length, composi-tion, and charge density) as well as the zipper domain (electrostatic charge), thereby fine-tuning the over-all properties of the gelation phase diagram. Recently, additional functionality has been imparted into leucine zipper hydrogels through the incorporation of folded globular proteins. For example, the inclu-sion of an alcohol dehydrogenase with aldo−keto reductase activity (AdhD) into a leucine zipper protein polymer led to a thermostable, self-assembling hydrogel with enzymatic activity (Wheeldon et al. 2008).

WW and proline-rich domains represent another example of associating peptides that have been used to design protein-engineered hydrogels. Numerous WW domains, so named for their conserved tryptophan (W) residues, have been identified in intracellular proteins and also derived computation-ally (Russ et al. 2005). WW domains bind to proline-rich sequences, which are divided into several dif-ferent classes with varying dissociation constants. The design of protein block copolymers containing multiple WW or proline-rich domains connected by hydrophilic peptide spacers enabled the formation of a mixing-induced, two-component hydrogel (Wong Po Foo et al. 2009). The large library of various WW and proline-rich domains allowed for modulation of the crosslinking strength, and hence hydro-gel viscoelastic properties, based on the binding affinity of the chosen domains. In addition, the use of

4-7 Engineered Protein Biomaterials

transient physical crosslinks to form the protein hydrogel resulted in a shear-thinning and self-healing biomaterial, which is required for injectable theraputic applications.

4.3.2 Structural Domains

In addition to the density of crosslinking sites, the mechanical properties of a protein-engineered biomaterial can also be controlled by including various structural peptide domains in the primary sequence. Elastomeric proteins contain domains that cause them to exhibit rubber-like elasticity, enabling them to undergo high levels of reversible deformation under high stress (Tamburro et al.

2010). Elastin and silk are elastomeric proteins that have been extensively studied and whose desirable mechanical properties have been incorporated into many different biomaterials. In addition to their structural properties, elastin-inspired polymers have been explored for use as injectable biomaterials and implantable scaffolds due to their biocompatibility and thermal sensitivity (Cappello et al. 1990).

Through a combination of protein sequence selection and spinning conditions, silk fibers have an out-standing combination of mechanical properties—high strength, elasticity, and resistance to compres-sion failure—that is highly desirable for biomaterials (Gosline et al. 1999). In addition, they have been found to have tunable degradation rates and to be biocompatible (Park et al. 2010). Attempting to harness these properties, researchers have succeeded in designing multiple versions of recombinant silk through expression in host systems, such as yeast, E. coli, and mammalian cells (Fahnestock and Bedzyk 1997, Asakura et al. 2003).

A recent addition to the library of structural domains included in protein-engineered biomateri-als is resilin. This protein enables many insects to fly, jump, and vocalize, both by storing energy in sound-producing organs and by constraining vibrations during flight. Natural resilin from locusts and dragonflies has demonstrated a remarkable fatigue lifetime and up to 92% resilience (ability to recover after deforming under applied stress) (Tamburro et al. 2010, Elvin et al. 2005). Resilin-derived peptide sequences were observed to have no stable secondary structure and instead underwent continuous inter-conversion between extended (poly-l-proline II) and folded (β-turn) conformations, allowing resilin to act as an entropic spring. The structural resilin domain has been incorporated into engineered pro-tein biomaterials combining multiple biofunctional domains, including the RGD ligand for cell bind-ing, a matrix metalloproteinase-sensitive sequence for proteolytic degradation, and a heparin-binding domain for the binding and controlled release of growth factors (Charati et al. 2009). The crosslinked material was found to be both highly elastic and to promote cell attachment and proliferation, making it an ideal candidate for mechanically demanding tissue engineering applications.

Another way to use proteins as structural domains is to harness their self-associative interactions to create specifically shaped nanostructures. For example, structures such as hollow cages may be used as drug or gene delivery materials (Uchida et al. 2007), while self-assembled compact structures, such as M13 bacteriophages, can be used to display a high density of a cell-binding peptide (Chung et al. 2010).

While the above examples utilized protein self-assembly to form naturally evolved structures, scientists can also mix and match various peptide domains to form novel self-assembling nanostructures. As an example, several rigid α-helical peptide domains that either dimerize or trimerize were fused together

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