Jun. 11, 2015

The Silk Road’s Turn Toward Biotechnology

by Julie Leibach

Click to enlarge images
This article is part of a spotlight on silk. Check out “The Medical Wonders of Worm Spit,” part of SciFri’s Macroscope video series, and this activity involving a silkworm cocoon dissection.
 
 
There’s a glass cabinet of curiosities that stands in the corner of a windowed room of the silklab at Tufts University in Boston. Among its novelties: a remote control airplane; half of a hard-boiled egg preserved in shimmery, mica-like material; a few cups reminiscent of Styrofoam ones; and several miniature skulls.
 
Reaching into the case, Fiorenzo Omenetto, a professor of biomedical engineering and physics and co-director of the lab, removes one of the coffee cup-sized skulls. “This is actually ‘mini Yorick’,” jokes Omenetto (his students call him “Fio”), referring to the character exhumed in Shakespeare’s Hamlet.
 
{"input":{"width":"285","photo":"cabinet","row":"5146","table":"DOCUMENT"}}
As disparate as this small skull and its curio mates may seem, they share a common thread: They were all fashioned, at least in part, from a protein found in silk, that sinewy material produced by the caterpillar of the silkworm moth, Bombyx mori.
 
In some cases, the objects are more kitschy than functional—side projects reflecting a Willy Wonka-esque creativity that thrives in Omenetto’s lab. Here, he and around a dozen students have been investigating how a silk protein called fibroin can be processed and fashioned into biodegradable materials and devices with a range of potential applications in medicine and beyond, from diagnostic imaging, to drug storage and delivery, to sensors for monitoring food quality.  
 
“You have an idea, you’re free to pursue it,” says Mark Brenckle, a Ph.D. student in Omenetto’s lab. “But any time you don’t have an idea, just walk into Fio’s office, and you’ll walk out five minutes later with 10.”
 
An old lab notebook of Omenetto’s, labeled “Stuff October 2005”—the year he joined Tufts—is testament to that fact. Brimming with notes, “it has a lot of ideas that seemed a bit crazy at the time but that certainly did end up coming true,” says Omenetto, whose office is a small box with a poster on the door that reads, “I am Italian and I cannot keep calm.”
 
The work spinning out of the lab is rooted in pioneering research done by fellow Tufts biomedical engineering professor David Kaplan. Nearly three decades ago, Kaplan began studying the properties and functions of silk fibroin as a naturally occurring polymer. His team now explores how the protein can be used in tissue engineering and regenerative medicine, among other medical applications. [For more on Kaplan’s work, watch “The Medical Wonders of Worm Spit.”]
 
After a decade of research, often in collaboration with Kaplan’s group, Omenetto sees silk fibroin as “an absolutely amazing bridge” between biology and technology in the push toward improved personalized medicine. “It’s not just another material,” he says. “It’s a collection of functions.”
 
{"input":{"width":490,"photo":"skulls","row":"5146","table":"DOCUMENT"}}
 
 
Silk was first woven into a textile some 5,000 years ago in China. Since then, with the advent of mass production, the material has become ubiquitous, “whether it’s in clothing, ties, bed sheets and pillow cases, and so on,” says Kaplan. “It’s very durable as a protein fabric, and that’s really why there’s such a large-scale production around the world of this protein.” Obtaining silkworm cocoons for research, therefore, is relatively easy and inexpensive, he says. Tufts has used cocoons from Japan, China, Thailand, and India, as well as South America. [For a cocoon dissection activity, click here.]
 
In hand, the whitish, egg-shaped capsules are small and soft, like felt. In the caterpillar, however, silk starts out as a liquid mixture of proteins, salt, and water stored in an organ called the silk gland. As the silkworm spins its cocoon, that concoction—extruded through the caterpillar’s mouth—transforms into a long, thin fiber that’s “durable against all kinds of environmental insults,” says Kaplan.
 
In order to process silk for research, the researchers must convert the fiber back to a liquid state, purified to contain only the fibroin protein. The process is relatively simple, involving: cutting cocoons into small pieces; boiling those in a weak alkaline solution; rinsing and drying the gossamer fiber that detaches (the fibroin); dissolving that using a salt solvent; and then running a dialysis in water to finally create an aqueous fibroin solution. From beginning to end, the method is relatively benign, and generates very little waste.
 
 
From that starting point of aqueous solution, silk fibroin then becomes “very tuneable,” as Kaplan puts it. In other words, it can be processed into multiple forms—from spongy scaffolds to support cell growth, to adhesive gels for holding damaged tissues together, to hardened structures for connecting fractured bone. Processing can be done using simply water at relatively low temperatures, which is not only more environmentally friendly, but enables the protein to be mixed with other biological components, such as cells that might deteriorate under conditions requiring harsher chemicals or extreme temperatures.
 
When it comes to implantation in the body, silk fibroin has several attractive properties. For instance, it’s biocompatible, meaning that it can be inserted in living tissue without initiating an inflammatory response or immune system attack. (In contrast, polyesters—another popular class of biopolymers used in medical devices—are more prone to inflammatory response, according to Kaplan.) Indeed, silk sutures have been FDA-approved for decades (and have been used medically for centuries), although “there were essentially no other medical devices being made from silk until we started about 15 years ago,” says Kaplan.
 
Silk fibroin is also biodegradable and bioresorbable—it will dissolve when placed in the body in a prescribed amount of time, on the order of hours to even years, based on the processing method used.
 
“I've never seen or found or worked with any other material from nature that offers the versatility and range of properties that you can achieve with this remarkable family of proteins,” says Kaplan.
 
And when Omenetto joined the team at Tufts, the researchers discovered yet another virtue: Silk’s utility as an optical material.
 
 
 
Omenetto has long had a fascination with light. As a kid growing up in Varese, Italy, his analytical chemist father would invite him to watch experiments involving flame spectroscopy, which uses a flame’s light to determine the amount of chemical element in a substance. “Controlled light and controlled fire—it doesn’t get any cooler than that,” says Omenetto, 48. The idea of harnessing light’s ethereal nature intrigued him. “I thought it was kind of black magic,” he says.
 
After completing his doctorate in applied physics (he also has a master’s in electrical engineering), Omenetto worked in the field of optics at Lost Alamos National Laboratory, investigating the way high intensity light from lasers propagates through materials such as optical fibers (a branch called nonlinear optics).
 
When he joined the biomedical engineering staff at Tufts, however, Kaplan wasn’t sure how Omenetto’s expertise would fit in. “I knew he was very dynamic, very smart, [had] great ideas,” says Kaplan, “but exactly where his niche would be in BME [biomedical engineering] was something to be determined.”
 
A fortuitous meeting in the hallway led to a revelation. Kaplan’s lab had been developing tissue-engineered corneal implants, which entailed growing corneal cells on a scaffold of clear and ultra-thin, silk-based films (the research is ongoing). The technique required stacking the films on top of each other—but for nutrients and oxygen to filter through the stacks to reach the growing cells, the layers required careful perforation.
 
It dawned on Kaplan that “[Omenetto] had these fancy lasers that could do all sorts of odd things with materials,” he recollects. Upon running into his colleague in the hall, Kaplan asked Omenetto to “poke some holes” in the scaffold.
 
Omenetto obliged, and was stunned at what he discovered. When he shone the laser on the thin silk film, it went straight through rather than scattering across the surface, as it would have with a rougher material. Omenetto found himself thinking, “Hey, this must be a very good optical material.”
 
Indeed, silk is transparent to light of all visible wavelengths, on par with most transparent plastics or glass. Second, it’s extremely smooth, with a reasonable refractive index, comparable to glass. What’s more, silk can be processed on the micro- and nano-scale to contain tiny geometric patterns designed to interact with light.
 
The epiphany set Omenetto’s research in motion. “We started thinking that if we could mix biological stuff in, it would make for very unusual optical devices,” he says.
 
{"input":{"width":490,"photo":"mirrors","row":"5146","table":"DOCUMENT"}}
 
A few years ago, Omenetto’s lab developed a prime example of a silk-based optical device: a sheet of what are essentially tiny mirrors—also called microprism arrays—designed to be fully implantable and resorbable in the body. The mirrors are made entirely of silk fibroin that’s been cast in a mold based on commercial reflector material. When light shines on them, micro-scale geometric patterns reflect it, just like a stop sign.
 
Reporting in Proceedings of the National Academy of Sciences (PNAS) in 2012, Omenetto’s team demonstrated several potential biomedical uses for these microprism arrays. In one in vitro experiment, they used imitation tissue (called phantoms) to demonstrate how the device could enhance tissue imaging. As a theoretical example, the idea is that if the mirrors were implanted in the body under a tumor and near-infrared light shone upon the area, they would reflect light in a way that created the contrast necessary to gauge the size of the growth.
 
In another set of experiments, the researchers loaded, or “doped,” a silk fibroin solution with a cancer-fighting drug called doxorubicin, and then molded that into mirrors. When they exposed the prisms to enzymes in vitro, the mirrors degraded—and the light that they reflected decreased. By monitoring the change in light reflectivity, they found that they could assess the rate of drug release.
 
While the mirrors are a proof-of-concept, their potential applications are still worth considering, according to Tiger Tao, lead author on the PNAS paper and a former post-doc and researcher in Omenetto’s lab. If someone suffers from a localized tumor, for instance, being able to administer targeted therapy and monitor drug release is crucial because “cancer-killing drugs, they’re very nasty,” says Tao. “You have to make sure the drug is released at a proper rate so that it mostly kills the tumors rather than surrounding good tissues.”
 
Further, unlike many drugs available today that must be refrigerated, silk is remarkably stable at room temperature, even when doped with drugs. “All these aspects together make this simple [mirror] device a more functional [one],” says Tao.
 
{"input":{"width":490,"photo":"cocoons","row":"5146","table":"DOCUMENT"}}
 
 
As part of their effort to add technological function to silk fibroin, Omenetto and his team have more recently been exploring silk’s ability to interface with electronics.
 
“There’s this emerging idea of taking biomaterials and polymers that are typically used in one context, then imparting some kind of interesting electronic functionality to them,” says Christopher Bettinger, an assistant professor of materials science and biomedical engineering at Carnegie Mellon University, who's familiar with the work at Tufts (he also co-authored a paper with Kaplan).
 
In the case of a silk-based electronic device, the key is that “the electronic materials, like the silk, have to be biocompatible and bioresorbable,” says John A. Rogers, a professor of materials science and engineering at the University of Illinois at Urbana-Champaign who has worked with Omenetto and Kaplan on several projects. Otherwise, “you need a second surgery to go in and fish out the hardware.”
 
To that end, a team including Rogers and Omenetto made strides this past November when they reported development of a wireless, remote-controlled, silk-based device referred to as a “magnesium heater,” and designed to kill a localized bacterial infection.
 
“This was actually the first [in vivo] demonstration of an implantable device that can be fully degradable with a controllable lifespan that also had some real functionality,” says Tao, also lead author on this study, published in PNAS. (See a diagram here.)
 
{"input":{"width":490,"photo":"heater","row":"5146","table":"DOCUMENT"}}
 
At about a quarter the size of a postage stamp, the invention consists of thin, transparent silk films sandwiched around what looks like a maze—the kind a kid might solve in an activity book, only tiny. In fact, the “maze” consists of a serpentine resistor and a power-receiving coil made from magnesium—an essential ion required by the body. Because magnesium degrades rather quickly in living tissue, the silk serves as a protective shield, designed to disintegrate at a slower rate.
 
To demonstrate its utility, the researchers inserted the device under the skin of mice infected with Staphylococcus aureus. Using a remote control, they signaled the magnesium coil to heat up the resistor, which in turn killed the bacteria. The team also tried doping the silk fibroin with a drug, as they had with the mirrors. They found that, in vitro, heat delivery triggered drug release and destroyed bacteria.
 
“The vision [for this device] would be that you don’t have to take pills on a regular schedule,” says Rogers. “Instead, all of the needed medicines are embedded in this platform, and then you just hit a wireless trigger to release those drugs.”
 
In addition, because the device works on the local level, it could potentially be inserted into surgical incisions before they’re sewn up. “There are probably 10-15 percent of patients who suffer from this post-surgical bacterial infection,” says Tao.
 
The researchers are currently building on this work, according to Rogers, and have submitted another paper that they hope will come out some time this summer. While it would ultimately take about seven years for a device like the heaters to make it to market, the concept has a few points in its favor, Rogers says.
 
For one, like silk in suture form, the antibiotics the team used are already FDA-approved. “The fact that these materials have been used for other kinds of implants gives one confidence that there are no intrinsic biocompatibility challenges with the materials that we’re using,” he says. “It’s hard to necessarily predict these things, but I think we’ve made a reasonable set of choices.”
 
Of course, the leap from lab bench to bedside entails other hurdles, too. Some surgeons might not see a need for a device that dissolves in living tissue, says Bettinger, and “in some sense, those are the customers that one would have to convince first in order to use this kind of new technology.”
 
{"input":{"width":490,"photo":"airplane","row":"5146","table":"DOCUMENT"}}
 
 
It’s tempting to envision silk fibroin as a wonder material, and in many ways, it is. For Omenetto’s part, the emphasis shouldn’t be on silk, per se, but on the effort to repurpose naturally derived materials into “smart” products with technological function and low environmental impact, be they biomedical or lifestyle-driven (think, wearable electronics). “Silk is certainly an excuse. [But] I think that it’s larger than that,” he says.
 
“I think there are going to be a lot of interesting things that will come out of co-opting these materials for technological applications, or the next generation of bioplastics, and specific interfaces at the micro- and the nano-scale that bring technology and biology together,” Omenetto says.
 
For instance, Bettinger is developing a battery-operated, ingestible therapeutic device consisting of natural polymers, including sugars and fats, and even cinnamon spice. Omenetto’s own lab has studied goat cashmere, which contains keratin, another biopolymer that has advantages over silk in that it interfaces better with some metals.
 
Still, the silk road is long, with miles left to explore. “Think about how all of these [silk] forms can be empowered by easy doping, by easy mixing of things that blink, things that live, things that bind oxygen, things that heal, etcetera,” says Omenetto. “The cookbook of technology is very expansive.”
 
 
 
*This article was updated on June 12, 2015, to reflect the following changes: When the silk fibroin is prepared, it must also go through a dialysis step and time in a centrifuge in order to become a usable aqueous solution. The honey-tinged, viscous solution originally mentioned is how the fibroin appears in the salt solution. Also, an earlier version stated that in the magnesium heater experiment, a smaller amount of heat was necessary to cause drug release than when the silk wasn't doped with drugs. It was a comparable amount of heat.
About Julie Leibach

Julie is the managing editor of ScienceFriday.com. She is a huge fan of sleep and chocolate. Follow her @julieleibach.

The views expressed are those of the author and are not necessarily those of Science Friday.

Science Friday® is produced by the Science Friday Initiative, a 501(c)(3) nonprofit organization.

Science Friday® and SciFri® are registered service marks of Science Friday, Inc. Site design by Pentagram; engineering by Mediapolis.

 

topics