Particle size matters


Studies fail to include basics for assessing toxicity

By Candace Stuart

Vicki Colvin has a question for colleagues who study nanoparticles and how they may affect people and the environment. “Exactly what do you mean by size?”

When chemists, toxicologists or other researchers report the dimensions of an engineered nanoparticle, are they measuring the core, the core plus a coating, or perhaps the core, a coating and attachments that help nanoparticles adhere to cells? What happens after exposure to water, or to blood?

“We want to know how particle size changes as it marches through the body,” Colvin said at a workshop designed to identify roadblocks to nanobiotech commercialization. Size, composition, shape and other characteristics help distinguish the scores of different engineered nanoparticles that exist today. They also help determine their wanted - and unwanted - properties. “Can I take a material that is active (potentially harmful) and make it safe?” she asked. “How can I engineer a safe nanoparticle?”

Colvin, a chemistry and chemical engineering professor and director of the Center for Biological and Environmental Nanotechnology at Rice University, is not alone in her quest. The federal agencies that may decide to impose environmental, health and workplace regulations on industry face a mishmash of toxicological data that often lacks basic information about nanoparticle size, surface area and other characteristics.

Rice University researchers Vicki Colvin and Mark Wiesner explore the two sides of nanotechnology: its use for remediation as well as its possible toxic effects. Photo by George Craig
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Toxicologists and other scientists studying nanomaterials say these gaps make it difficult if not impossible to compare studies and get an accurate picture of how nanoparticles interact with the body.

Reporting basics like size will go a long way toward ensuring that regulations are based on sound rather than spotty science, they say. Scientists will be able to see the relationships between a nanoparticle’s size or surface area or charge, for instance, and how it behaves in the body to predict which traits could be harmful. That knowledge may help them design benign nanoparticles.

“It was kind of where dioxins and PCBs were in the ’60s,” said Nigel Walker, a staff scientist with the National Toxicology Program (NTP) of his initiation into nanotechnology about three years ago. NTP, part of the National Institutes of Health’s environmental sciences division, has launched a program to evaluate potential health hazards of nanomaterials. Dioxins, a byproduct of combustion processes, and polychlorinated biphenyls, an industrial chemical, were found to be cancer-causing pollutants that required costly cleanups once their toxicity was discovered.

“We introduced a technology without understanding the implications and then spent 30 years trying to eliminate or reduce the risk,” Walker said. He recognized that with nanotechnology he and other scientists had an opportunity to spot troublesome nanoparticles early in the commercial process, before they cause damage. “Now we can make sure we can prevent that. We can choose the kinds of experiments that reduce the risk. We can be at the forefront.”

Their size makes nanoparticles promising candidates for medical applications. They are small enough to fit within cells and also can roam undetected by biological sentries such as the blood-brain barrier or the liver, according to Scott McNeil, director of the National Cancer Institute’s Nanotechnology Characterization Laboratory. As coordinator of pre-clinical characterization of nanomaterials, McNeil is helping the NCI in its goal of developing nano-based therapies and diagnostics for cancer.

His team has already begun tests on gold nanoparticles, liposomes, dendrimers and buckyballs. Each nanoparticle offers benefits: Branch-shaped dendrimers and spherical liposomes can transport drugs into cells. Contrast agents can be put in the hollow centers of buckyballs for tumor imaging. Gold particles known as nanoshells can attach to cancer cells, and when exposed to harmless near ultraviolet light, heat up and kill the cells.

Preliminary research on gold nanoparticles suggests that nanoparticles grow larger in the presence of plasma. Researchers consider the results a trend until their studies undergo further review. Image courtesy of the Nanotechnology Characterization Laboratory
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“These can leach into tumors because they are smaller than the pores of the blood vessel wall,” McNeil said. Nanoparticles are also smaller than the filter mechanisms in the spleen and liver that capture and eliminate other foreign matter. That leaves nanoparticles free to circulate until they find their cancer target.

Nanoparticles pose a risk, though, if they are or become toxic and the body’s defense systems can’t detect and eliminate them, McNeil and Walker pointed out. For medical applications - where tissue is deliberately exposed to nanoparticles - it is critical to understand how cells react to the presence of various types and forms of nanoparticles during various stages of exposure. Unintended exposure through the skin, lungs and other pathways also needs to be considered.

“Size will be a critical component for toxicology,” Walker said. Studies on ultrafine particles, which are typically a byproduct of combustion, suggest a link between particle size and toxicity, for instance. “But size is contextual. How do you report size? What kind of methodology do you use? What is the tool?”

The Nanotechnology Characterization Laboratory is collaborating with the Food and Drug Administration and the National Institute of Standards and Technology to find methods for analyzing nanomaterials in various stages: before exposure to any biological environment, exposure in test tubes and other in vitro environments, and exposure within living organisms, or in vivo environments. They accept applications for materials from manufacturers on a quarterly basis, but will only take materials that can be produced in sufficient quantities.

“We don’t accept material for characterization unless they can produce a gram of material. We don’t want to have multiple industrial batches,” McNeil said. Batches can vary in purity, for instance. “We want to make sure we have the same stuff in the lab as in the animal.”

McNeil’s team follows a methodical system to plot the physical attributes of each type of nanoparticle. But nanoparticles rarely are used in their natural, or “naked,” form. Many nanomaterials that function in the dry world of chemistry need to be “dressed” to work in the wet world of biology. They also often have antibodies or other biomolecules attached to their surface that complement molecules on a cancer cell’s surface. The attachments help the nanoparticles latch onto target tumor cells.

“We attempt to have a baseline characterization so we can have some familiarity with that category of nanoparticle,” McNeil said. Each time it is dressed, or functionalized, it is studied again. “Each one would be unique in its behavior. Functionalizing it changes its properties.”

Designers can use the ability to alter properties in their favor. A nanoparticle’s surface charge can make it less biocompatible, for instance. Adding a coating that neutralizes charge may solve the problem, though. In what McNeil characterizes as a possible trend, his team noticed naked gold nanoparticles get larger when placed in plasma. To better understand gold nanoparticles’ blood-contact properties, they plan to add molecules that could stop proteins from absorbing on the surface. That also may be a way to keep the gold nanoparticles from growing larger.

The National Toxicology Program is studying buckyballs and carbon nanotubes, semiconductor nanocrystals called quantum dots and metal oxides such as the sunscreen ingredient titanium dioxide. It is developing a program based on dendrimers, too. NTP, which agreed to take on the initiative at Colvin’s request, allocated between $1.5 million and $2 million in the past year for the program, Walker said.

Veeco’s BioScope II allows scientists to image molecular features within living systems. Photo courtesy of Veeco Instruments
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Walker said the NTP is more or less starting from scratch after scouring the toxicology literature and finding it lacking. But accurately measuring nanoparticles in their various states may prove difficult. Microscopy tools for measuring an electron-dense nanoparticle may not be as suitable for gauging less electron-dense coatings or attachments. Tools using probes may compress flexible structures.

Craig Prater, a fellow at Veeco Instruments who has been integral in the commercialization of several of its microscopy products, said today’s tools can adequately measure nanoparticle size, even in the wet world of biology. Prater helped launch Veeco’s nanobio atomic force microscope, the BioScope. The BioScope II, introduced in December, can be used to image molecules within living cells.

He’s also involved in a project with Dow Chemical Co. to develop ways to better understand nanoparticles’ structure-function relationships. The goal of the Veeco-Dow partnership is to provide a predictive mechanism that will allow manufacturers to efficiently design nanoparticles that perform exactly as desired.

“If we can understand the structure-function and surface chemistry connection to toxicity, that will help us accelerate the design of consistently safe nanoparticles,” Prater said. “All of the chemical companies and scientists take that responsibility seriously.”

But reliable predictions depend on sound data, and scientists like Walker, McNeil and Colvin warn that, for the most part, good fundamental information is not available now. The haphazard reporting of basic characteristics like size threatens to hamper commercialization. “We need to do a better job of describing material,” Walker said. “This will be a roadblock.”

In an effort to get consistency in nanoparticle reporting, Walker contacted funding agencies and editors of key scientific journals and asked that grants or acceptance of a paper be contingent on reporting basic information such as size and methodology. Colvin has been prodding the academic research community to agree to some accepted norms. McNeil hopes to develop voluntary standards for industries and toxicologists based on a consensus process.

It’s the chance to avoid another dioxin or PCB incident.

“If we could provide the structure-activity tools,” Walker said, “then we could say (to nanoparticle designers), ‘You really don’t want to go there with this one.’ They could make an informed decision. The further we can get to providing that assessment upfront, the better it will be for everybody.”

Online sources of information

everal organizations offer searchable databases and inventories on nanoparticles and their health and environmental implications. Here are a few:

International Council on Nanotechnology

U.S. National Institute for Occupational Health and Safety

European Union’s NanoSafe project

Wilson Center’s Project on Emerging Nanotechnologies