Issue Date: July 21, 2008
From Diseases To Devices
IMAGINE ONE DAY placing a call on a cellular phone made with a plastic that contains disease-causing proteins.
Misfolded protein aggregates called amyloid fibrils are the proverbial bad guys implicated in such illnesses as diabetes, Alzheimer's disease, and bovine spongiform encephalopathy (mad cow disease). Because of their close association with disease, these clumps of insoluble proteins generally fall into the realm of biomedical research.
But the greater-than-steel strength of amyloid fibrils—combined with their flexibility, versatility, and ability to self-assemble—has also begun to pique growing interest in their potential as advanced nanofiber materials. Although researchers once saw amyloid fibrils primarily as harbingers of disease and decay, they have begun to envision useful possibilities for them. They are now using fibrils to design new types of conducting nanowires, bioactive ligands, and biodegradable materials, among other potential applications.
Until recently, the only known amyloid fibrils were either linked to disease states or found naturally in biological structures, such as in the curli proteins in the extracellular matrix of biofilm bacteria. Such naturally occurring fibrils are certainly important, but they don't provide the range of structures and functions needed for engineered nanostructures, so the idea of amyloid-based nanomaterials had remained dormant for some time.
In 2000, however, the laboratory team of Christopher M. Dobson, professor of chemistry at the University of Cambridge, synthesized an amyloid fibril from two different kinds of peptides, thereby showing that such a synthesis was possible. Dobson's group also tagged the peptides with fluorescent markers, which ended up on the outside of the resulting fibrils. By adding fluorescent function without compromising the structure of the fibril, Dobson's group made a crucial leap toward the development of amyloid nanomaterials.
When examined by electron microscopy, amyloid fibrils appear as aggregates of single unbranched fibers, with an aggregate diameter of several nanometers and a length extending 1 µm or more. Every one of these natural amyloid fibrils starts as misfolded globular proteins. No one knows exactly what triggers these proteins to misfold and aggregate, but researchers have successfully aggregated proteins experimentally by stressing them through changes in pH, solvent, and temperature. Stress the proteins just enough to encourage slight unfolding, and they may begin to aggregate and form fibrils.
Virtually all globular proteins can self-assemble into amyloid fibrils, although they don't all do so with the same efficiency. Proteins containing large numbers of amino acids with aromatic or hydrophobic side chains tend to aggregate with ease. A slightly unfolded, stressed protein can potentially expose these side chains to the aqueous solution of the cell. To shield these exposed side chains as much as possible, the stressed protein may then aggregate into a series of "cross-β-sheets," flat ribbons of β-strands that run perpendicular to the long axes of the fibrils.
Sally L. Gras, a researcher in nano- and biochemical engineering at the University of Melbourne, in Australia, explains that an amyloid fibril is like a stack of cross-β-sheet Lego pieces. The cross-β-sheets interact via noncovalent interactions, such as hydrogen bonding, π-π stacking, and Coulomb attraction, ultimately forming a degradation-resistant fibril.
THIS TYPE of fibril resists not only degradation but also breakage and shearing. Many compare the amyloid fibril to spider silk, a fiber composed of self-assembling peptides. Both are remarkably strong. However, "the only thing that can make spider silk is spiders," nanoscientist Mark E. Welland of the University of Cambridge says, whereas amyloid fibrils form spontaneously.
Much of the promise of amyloid-based nanomaterials arises from their ability to self-assemble from component molecules. Think of a box of parts morphing into a chair, fully assembled and intact. Instead of putting your chair together one twist of the allen wrench at a time, you spread the parts on the floor and simply wait. The chair builds itself from the bottom up, each part traveling unaided to its final destination. Synthesizing amyloid fibrils is even simpler because all of the "parts" are the same. Each fibril consists of structures derived from thousands of identical proteins. Encourage globular proteins to unfold slightly, and these otherwise soluble proteins may begin to self-assemble into an insoluble amyloid fibril.
Of course, researchers don't want self-assembly to take place completely at random. To be a valuable nanomaterial, one that can be reliably synthesized, scientists must to be able to exert some control over how and when it assembles. That's why researchers have developed a variety of triggers that initiate fibril formation under specific circumstances. Most commonly, a small change in pH can alter interactions of charged side chains, exposing hydrophobic groups and encouraging fibril formation. Enzymes can also be used to induce two small precursor peptides to combine and form a single self-assembling fibril.
A key advantage of de novo syntheses of amyloid fibrils is that they allow biochemists to choose the building blocks of these fibrils. Unlike the naturally occurring amyloids found in humans, those made in the lab can include amino acids not otherwise found in nature. For example, although evolution has selected primarily for left-handed amino acid enantiomers, right-handed ones can be used in fibril synthesis. "There are a huge number of combinations of things you can do," Gras says.
Welland agrees. The ability to easily synthesize amyloid fibrils in the lab adds to their appeal. To make these fibrils, he says, "all you need is a bucket and some simple chemistry."
For example, Gras, Dobson, and physicist Cait E. MacPhee of the University of Edinburgh, in Scotland, used a transthyretin peptide as the building block for a synthetic amyloid fibril. They then made a second synthetic fibril from transthyretin to which they added a fibronectin-derived arginine-glycine-aspartic acid (RGD) motif to serve as a model bioactive ligand. Fibrils bearing the RGD motif bound to cells during an assay, whereas control fibrils without the RGD motif didn't bind, indicating that the motif added functionality and bioactivity to the otherwise nonreactive amyloid fibrils (Biomaterials, DOI:10.1016/j.biomaterials.2007.11.028).
RESEARCHERS ARE already eyeing such fibril-borne bioactive ligands as a means of immobilizing enzymes. Immobilization can make enzymes more thermostable, enhance their activity, and increase the efficiency of large-scale reactions. "You don't want an enzyme to float about and come out with your product," Gras noted. By immobilizing it, "you can do the same reaction on a microfluidic chip instead of in a big tank and concentrate it in one area," she adds.
Researchers have also begun to examine the biocompatibility of amyloid nanomaterials. Amyloid fibrils are believed to be more biocompatible than other bionanomaterials because of their protein origin, and their biological nature may also help make amyloid biomaterials more stable under physiological conditions, Gras says.
The potential biocompatibility of amyloid fibrils—combined with their strength and rigidity, which can be downright metallic—would make them good candidates for implanted devices. In fact, their strength almost exactly matches that of bone and teeth. Gras notes, however, that it remains unclear whether these fibrils are toxic in the body.
Using amyloid fibrils in electronics design is also intriguing. For example, a group led by Ehud Gazit, professor of molecular microbiology and nanotechnology at Israel's Tel Aviv University, recently used analogs of amyloid fibrils and other bionanostructures to create a conducting biomaterial. The researchers created a hollow, self-assembling diphenylalanine-based synthetic tubular fibril and used it to cast a silver nanowire from silver nanoparticles. Dissolving the diphenylalanine nanotube with proteinase K exposed the silver nanowire within. Such metallic nanowires may be used in next-generation computers, batteries, and light-emitting diodes.
THE ABILITY to make a self-assembling peptide nanotube fibril using only diphenylalanine could prove to be significant because such a dipeptide is considerably cheaper than the larger peptides traditionally used to construct self-assembled fibrils. Gazit points to one dipeptide, aspartame, marketed as the artificial sweetener Equal, that is synthesized for a few cents per gram. The key to using nanowires and other items created with self-assembling peptides on a large scale is cost, Gazit says. "It's a delicate play between demand and development," he adds.
Nevertheless, a desire for amyloid-based nanomaterials is indeed beginning to arise in industry. The cellular telephone company Nokia has begun design on a partly biodegradable and presumably environmentally more benign cell phone dubbed Morph. Prototype design began with a collaboration between Nokia and the Nanoscience Research Centre at the University of Cambridge, headed by Welland. Welland's team combined amyloid fibrils with other plastics on the market to create a composite material. They then used this material to construct a cell phone that is sturdy and yet partially biodegradable.
"By themselves, these proteins would be just a mat," Welland says. "But mix them in with a plastic, and the plastic takes on the strength of the proteins." However, he adds, in time the fibrils "just dissolve away." This would give Morph "a considerable environmental advantage," he concludes.
Prototypes such as the Morph phone appear to be only the beginning of many types of amyloid fibril devices and applications that might be developed. With the push for smaller and smaller transitors for computer circuits, as well as more sophisticated medical devices for implants, amyloid fibrils may be able to start filling these needs. Some researchers also say that fibril-based light-harvesting devices might be possible. Gras believes that while the potential of amyloid-based materials may not be fully realized in the immediate future, there will be "a lot of exciting developments" in the next 12 months.
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