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Functional biomaterials


Summary


Polypeptides represent an important segment of the extracellular matrix (ECM), which is required for cell and tissue growth. We present an extension of our idea of using coiled-coil segments as crosslinking segments to graft additional properties to the polypeptides that constitute the ECM. The idea was to create new functional biomaterial composed of elastin-like segments non-covalently cross-linked by means of coiled-coils which can be used not only for culturing cells in vitro but potentially also for tissue repair and other tissue engineering and medical applications. The new biomaterial would be improved by containing different functional domains covalently linked to the polypeptides of the cell matrix giving the material new functions such as preventing growth of pathogens, stimulating growth of cells, tissues and organs, supporting cell reprogramming etc. Various functional domains could also be incorporated into the biomaterial after its initial assembly through coiled-coil interactions with heterodimeric coiled-coil forming segments incorporated into the polymer like a “molecular Velcro”. We also suggest further improvements of this biomaterial involving the potentials for the regulation of its assembly/disassembly through heterodimeric coiled-coil interaction between constituents and disassembling peptide. Experimentally we designed and confirmed the expression of several functional polypeptides containing coiled-coil segments and elastin-like segments which will be used for further research on this field. Experimental verification of design is under progress, since we focused our efforts on the subprojects with more defined assembly of polypeptide nanostructures. We feel however that the proposed idea suggests a promising approach for manufacturing biomaterials with versatile functional properties with potential use in biotechnological, medical and pharmaceutical applications.


The idea and approach


Bioactive materials and their design technology enable an extensive range of applications for the medicine and related fields. Numerous biopolymers have been developed as scaffolds supporting growth of cells and tissues. Rationally designed polypeptides offer a promising way for constructing biomaterials. For instance, self-assembling and self-complementary amphiphilic peptides have already been investigated; most extensively among those were beta-sheet and fiber forming peptides (Stupp et al., 2005; Holmes et al., 2000).

What could be an advantageous environment for the mammalian cell growth? This question led us to design elastin-based self-assembling polypeptides, which could form new polypeptide material, which mimics extracellular matrix, which is the natural environment of different eucaryotic cells and provides different cell-growth supporting and protecting functions. Namely, the significant stability of elastin and elastin-related polymers and their tendency for self-aggregation make them ideal compounds for the development of synthetic nanomaterials.

The precursor of stable, cross-linked elastin is tropoelastin (Figure 1) which is composed of:

• hydrophobic domains which contain repetitive sequences, which provide elastomeric properties and can coacervate through temperature driven phenomenon,

• hydrophilic domains which contain residues important for cross-linking into stable polymer elastin (Bellingham et al., 2003).


Three hydrophobic domains flanking two cross-linking domains are sufficient to achieve self-assembly of elastin peptides (Figure 1).


Figure 1: Elastin peptides are aligned during coacervation of hydrophobic domains (square planar structures with spherical structures representing hydrophobic side chains) and can be therefore covalently cross-linked through lysine residues on hydrophilic domains (cylinders) (Keeley et al., 2002).

Elastin-like materials containing hydrophobic repetitive sequences have already been prepared (Lee et al., 2001), but to employ them as resilient biomaterials, the polypeptide chains have to be covalently crosslinked. We came to the idea that elastin-like segments (consisted of repetitive hydrophobic domains of elastin) could be cross-linked by strong noncovalent interactions by introduction of coiled-coil forming segments. In addition to coiled-coil segments we can also add other biologically active segments (Figure 2) such as functional domains for inhibition of bacterial growth or mammalian growth factors. Since the functional parts would be covalently incorporated into the scaffold, there would be no need to add them to cells in soluble form, which would also increase their local concentration. Particularly appealing idea seems to be to add functional segments, which have a heterodimeric coiled-coil appendix, which allows them to attach from the solution to the polypeptide matrix through coiled-coil interactions. This would allow addition of a cocktail of different functional elements that could be tailored individually to specific cell or tissue type and desired biological activity.


Figure 2: Schematic presentation of the polypeptide biomaterial composed of elastin-like segments and coiled-coil segments. Coiled-coil segments oligomerize and connect individual polypeptide chains of the biomaterial polypeptide scaffold. Functional polypeptide domains can be either incorporated into the polypeptide chain as part of the fusion protein or can be linked to the scaffold by interactions between coiled-coil segments of the scaffold and coiled-coil segment fused to the functional polypeptide.

An additional feature of such material would be the ability to regulate its assembly by mixing two different polypeptides where the coiled-coil segments of one polypeptide material component could heterooligomerize with the coiled-coil segments of the second polypeptide material component (Figure 3), triggering polymerization only in the presence of both components.


Figure 3: Schematic presentation of the regulated assembly of two polypeptides material components. One soluble polypeptide containing a coiled-coil segment capable of heterooligomerization remains soluble. Assembly can be the initiated only after the addition of the second polypeptide component.

This design also provides the ability to regulate the disassembly of the material by the addition of one the peptides comprising one component of the coiled-coil segments pair which is present in the biomaterial (Figure 4). Therefore we could gently disassemble polypeptide matrix without having to resort to harsh changes of physical conditions, such as temperature, osmotic shock, ionic strength or enzymatic digestion for cell recovery.


Figure 4: Schematic presentation of the disassembly of the polypeptide biomaterial after the addition of the peptide comprising coiled-coil segment (broken line ellipse), which is also present in the polypeptide material. The added coiled-coil segment competes with coiled-coil pairing of the polypeptide material due to its ability to bind to one of the coiled-coil segments of the polypeptide material. This approach can also be used to release the compounds immobilized to the polypeptide matrix through coiled-coil segment.



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