High Noon for Nano
Nanomaterials must earn their stripes
By Nick Houtman
Along a four-lane highway east of Corvallis, Oregon State University's Sinnhuber Aquatic Research Laboratory sits adjacent to rows of blueberries, hazelnut trees and hops on trellises. Nearby fields produce grass seed and edible crops such as pumpkin, peas, sweet corn and broccoli.
Amid the valley's agricultural bounty, researchers at the Sinnhuber lab have another growing industry in mind these days: nanotechnology. Manufacturers now promote more than 600 products for their "nano" qualities, from tougher glass and automobile coatings to fabrics, toothpaste and odor-fighting socks. Key to these innovations are engineered molecules such as zinc-oxide particles (used in sunscreens and as crystals in electronic devices, solar cells and lasers), carbon-based "bucky balls" and "fullerenes" (both named for visionary architect Buckminster Fuller) and semiconducting crystals known as "quantum dots" (according to Oregon Quarterly, Invitrogen in Eugene is the nation's leading manufacturer of quantum dots). The flow of new materials and methods for making them shows no signs of slowing.
Just as the growing nano-economy is a logical consequence of this new technology, so are questions about the impacts of nanomaterials on the environment and human health. These materials could contribute to a more sustainable economy through increased efficiency, greater product effectiveness and less waste, but their proliferation leads to a simple question: Are they safe? And if some nanomaterials harm developing animals, how does that harm occur and what specific nanomaterial characteristics cause the harm? Important clues are starting to come from common aquarium fish being raised at Sinnhuber.
Zebrafish Are Key
Inside the lab, students and technicians maintain more than 1,200 clear plastic aquariums, home to 10,000 zebrafish (Danio rerio) that may be key to the future of the nano-economy. A research team led by Associate Professor Robert Tanguay in the Department of Environmental and Molecular Toxicology has been exposing zebrafish embryos to more than 150 types of nanoparticles from nearly 20 different laboratories and companies in the United States and Europe. The resulting assessment record on the biological effects of nanomaterials may be the largest of its type, he says.
Zebrafish provide an ideal whole-animal system for rapid assessment, Tanguay explains, because they reproduce quickly and are easy to raise. Since embryos are transparent, developmental abnormalities can be readily observed through simple microscopy (see a microscope video of a regenerating zebrafish tailfin). Moreover, the zebrafish genome, like the human genome, has been fully sequenced, so researchers can pinpoint changes at the molecular level.
"At this stage of the game, we're looking at hundreds and hundreds of different nanoparticles and asking questions: What happens when nanoparticles are present during early embryonic development? Does development proceed normally or is it adversely affected?" says Tanguay. "Ultimately we want to explain how a given nanomaterial produces given responses in fellow vertebrates, but that's not the point right now. The focus right now is on gaining information about the materials themselves to help understand their specific properties that produce biological responses."
Zebrafish at the embryonic stage, he adds, provide a broad net that can capture and amplify the developmental consequences of nanomaterial exposure (click here to see a larger image). "It's like putting up a big solar panel where we're trying to capture all of the sunbeams. If you get an adverse response in a developing organism, you've hit something that you shouldn't have hit. You've blocked something that you shouldn't have blocked. The zebrafish model is also ideally suited to identify what was hit or blocked."
Timing Is Everything
The consequences show up with exquisite precision through the process of development. That's because embryos use most if not all of their genome to direct the normal construction of the body. "Pretty much every gene product that will ever be used in the lifespan of the organism is being used in some period of time during development," says Tanguay.
And if an embryo is exposed to a toxic agent at just the right time, the results will become apparent later in life — bodies that are malformed or organs (eyes, kidneys, livers, hearts etc.) that don't work properly. Moreover, such problems are irreversible. "If you screw up the developmental plan, you're not going to go back and redo it. If you have one eye, you're not going to go back and grow the second one. That ship has sailed," says Tanguay.
The researchers' goal is to identify those nanomaterial characteristics that product engineers can use safely, i.e., those that have no impact on the developing embryo. Conversely engineers can avoid those that cause trouble. "We're trying to help the development of this industry, not to hinder it," Tanguay says.
He and his team have found that most nanomaterials have no effect on zebrafish development. However, in response to the question, "Are they safe?" the answer may be, "It depends." In the January 2008 edition of the Journal of Toxicology and Applied Pharmacology, they reported that fullerenes (C60, spherical clusters of 60 carbon atoms) can cause oxidative stress, resulting in embryo death, fin malformations and other abnormalities. Former master's student Crystal Usenko was the lead author with post-doctoral scientist Stacey Harper and Tanguay.
Among the variables they tested was exposure to light. They placed zebrafish embryos in solutions with varying concentrations of fullerenes and let some develop in light and others in darkness. They found that when fullerene-exposed embryos developed in light, they experienced more oxidative stress and cell mortality.
Ironically, that kind of toxicity suggests that fullerenes could play a useful role in cancer treatment. By designing fullerenes with the ability to seek out and attach to tumor cells, physicians could expose them to light and thus spur their cell-killing properties. And just to complicate things, the researchers also showed that adding other molecules to fullerenes could help protect cells from such oxidative stress.
A Global Reputation
This kind of knowledge is crucial to product development and is just now emerging from research by Tanguay and others at OSU. In collaboration with Qilin Li of Rice University, OSU Associate Professor Alan Bakalinsky in the Department of Food Science and Technology is addressing interactions between fullerenes and yeast cells. They are aiming at one of the critical unknowns in nanoscience, the interaction of cell machinery with nanomaterials.
"We're trying to identify specific shapes and structures in manufactured nanoparticles that might cause damage to cells," Bakalinsky told writer Aimee Brown of Oregon's Agricultural Progress magazine recently. "If we can determine which shapes and structures are most dangerous to cell function, it should be possible to design materials to avoid those shapes and to minimize the risk of cell damage."
Both Tanguay and Bakalinsky have received support for their work from the United States Environmental Protection Agency (EPA). In addition, the Oregon Nanoscience and Microtechnologies Institute (ONAMI) is facilitating collaboration between OSU and University of Oregon researchers who specialize in "green nanoscience" through the Safer Nanoscience and Nanomanufacturing Initiative directed by Professor Jim Hutchison at UO.
If you don't believe that the state of Oregon is earning a global reputation in sustainable nanotechnology, just ask Stacey Harper about her trip to Denmark last January. Harper is a former U.S. EPA researcher and a post-doctoral scientist in Tanguay's lab. "I was one of six invited international speakers at the Interdisciplinary Nanoscience Center," she says. "I was there with Michael Graetzel, the guy who developed the really hot new solar cell, and the director of the Max Planck Institute (a leading research center in Germany). We really have made a name for Oregon. Under the Safer Nanomaterials and Nanomanufacturing Initiative umbrella of ONAMI, we are leading this research internationally."
Harper and Tanguay are also assembling a knowledge system to evaluate nanomaterials and to provide a basis for decisions by researchers, product engineers and government agencies. This Nanomaterial-Biological Interactions database will incorporate results from experiments with zebrafish, yeast, mice, fruit flies, human cell cultures and other model systems used in medical research. The goal is to create a freely available, practical decision-making system, reflecting scientific judgments on the relative importance of different kinds of biological consequences.
A library of nanomaterial properties now maintained by the National Institute of Occupational Safety and Health may become part of the database as well.
Harper is seeking expert opinions on data evaluation through the International Organization for Standardization, a global network headquartered in Geneva, Switzerland. With colleagues from government and industry, Harper is participating in an international task group on nanotechnology and sustainability.
"We'll be embedded in that effort heavily," she says. "No one else is taking it from the perspective of ONAMI, especially the Safer Nanomaterials and Nanomanufacturing Initiative, which has really led the way for sustainable development of nanotechnology. There's no reason not to design nanomaterials to be high performance and safe at the same time."