Toxicology of the Tiny
The race to know how nanoparticles affect living things is on, even as the use of those particles is increasing exponentially.
Already incorporated into consumer products ranging from baseball bats and clothing to sunscreens and toothpaste, engineered nanoparticles — ENPs — hold great promise in such areas as energy, pollution remediation, medicine and materials science. The nanotechnology industry is projected to be worth $1 trillion by 2015.
It is all made possible by the peculiar properties of nanoparticles, which are defined as having at least one dimension measuring 100 nanometers or less (a nanometer being one-billionth of a meter, or about one one-hundred-thousandth the width of a human hair).
As the number of new nanoparticles rapidly increases, they likely will be released in greater number and diversity into the environment — when the particles are fabricated, during the manufacture of nano-enabled products, throughout the useful life of those products, and at end of life as they find their way into landfills, incinerators and, potentially, soil, water and air.
"The environment is not just a destination for nanoparticles; it transforms them," says microbiologist Patricia Holden. "Pollutants move and change, with possible direct effects on the quality of water, food and air. These environmental concerns are human concerns."
Holden, a professor of microbiology at the Bren School of Environmental Science & Management at the University of California, Santa Barbara, and a lead scientist at the UC Center for Environmental Implications of Nanotechnology, was principal investigator on one study that provided evidence for what Holden refers to as a "nanoparticle effect" — a substance having increased toxicity at the nanoscale than it has in bulk form.
In Holden's study, she exposed bacteria to cadmium ions — well known as a toxin — and cadmium nanoparticles (also known as quantum dots). Using electron microscopy to compare their toxicity, she found that at low concentrations, cadmium ions and ENPs similarly inhibited bacterial growth, but at higher concentrations, ENPs were more toxic than cadmium ions. Further, while the quantum dots were highly reactive outside the cells, upon entering the cells they became even more reactive and destructive.
While the ions gathered at the outer edge of the membrane and remained there, leaving the cell intact, the ENPs entered the cell and accumulated, reaching concentrations far in excess of those outside the cells and destroying the cellular structure.
"We don't expect bacteria to readily take up engineered nanomaterials because they are too large to pass through the membrane," explains Holden. "But quantum dots damaged the membranes, enabling the particles to pass into the cells, where they were potent toxicants. Now that we know these nanoparticles have a specific and profound effect — a 'nanoparticle effect' — on bacteria, our next goal is to determine why.
"Understanding that can make it possible to 'design out' negative effects, which is key to the environmentally safe use of nanomaterials."
With nanotechnology itself a relatively young science, research on nanotoxicology is even younger. Nonetheless, according to Kristen Kulinowski, director of the International Council on Nanotechology at Rice University, roughly 3,600 papers dealing with some aspect of nanotoxicology have been published to date.
Scientists know quite a bit about ENPs' unique characteristics and abilities, and, according to Kulinowski, "We have pretty significant control over them, so there's enormous flexibility in what we can make." But they know less about questions such as whether and to what degree individual particles are toxic, why and under what conditions and concentrations toxicity occurs, and how long nanoparticles endure in the environment.
"Do bacteria break them down, or do ENPs persist, perhaps as toxicants to the bacteria that would otherwise break them down?" asks Holden. "Nanoparticles exist in nature, but we are making a lot more of them than occur naturally, and we are therefore enabling a level of dispersal that also would not occur naturally."
"Scale-up is accelerating," says Kulinowski, "but not necessarily in terms of new types of nanoparticles. And I cannot point you to a source that has an authoritative number for the total amount of nanoparticles that are out there. But the real question is, 'However many are out there, is that a problem? Are we creating enough of these materials to get into the environment?'"
And what happens if they do enter the environment? The race is on to find out.
"The field is changing so rapidly and the landscape of different particles is nearly infinite," says Holden, "We need to rationally but rapidly approach this new field to deliver guidance into the future for environmentally responsible synthesis of nanoparticles."
To that end, several other research groups have also conducted experiments designed to assess ENP toxicity to cells. A common theme among the various efforts is their examination of what has been called the "nano Trojan horse," a process by which nanoparticles "smuggle" toxic ions into cells by defeating cellular defenses that prevent the ions from entering on their own.
A group in Switzerland compared the toxicity of seven industrially important nanoparticles with that of the non-nano forms of the same materials.
One of their findings concerned uncoated iron oxide, which, the researchers noted in their paper, "has been repeatedly proposed for medical treatments such as magnetic drug targeting systems or as a contrast agent in magnetic resonance imaging." For this presumably safe item they found an ENP-specific toxicological mechanism.
Another group studying the effects of silver nanoparticles on bacteria at the University of Missouri found that the ENPs seemed to alter or inhibit certain defense functions that protect organisms from toxicants. The authors suggest that the particles may act by "directly reacting with the cell membrane to allow a large number of the silver atoms to attack or easily enter the cells."
They add, "The mechanisms by which silver nanoparticles kill microorganisms are … largely unknown, and the mode of antimicrobial action by nanosilver is not clear." The group found evidence that particles having dimensions of less than 10 nanometers "may enter the cell directly to inhibit microbial growth."
"Particularly with respect to silver," says Kulinowski, "there is some thinking that nanoparticles are acting as reservoirs for silver ions, and most organisms can't evolve enough defense to protect themselves from the ions. The nanoparticle may be the ideal delivery vehicle. Or nanoparticles may be using a different mechanism to act on cells than ions do."
While killing "microbes" is often seen as a positive, these experiments are to test the toxicity of ENPs on living organisms; bacteria or microbes are a good starting point because they are reasonably simple. The thinking is that if they have such a profound effect on these single-cell organisms, what will they do to a complex organism?
How nanoparticles behave in a liquid is another important aspect of these recent projects. Holden explains that the toxicity in nanoparticles engineered from toxic metals may not always be a novel new threat by the particles but a simple outcome of dissolution — that is, toxic levels of free ions are released when the nanoparticles dissolve.
This was borne out at the Centre for Environmental Contaminants Research in Australia, where scientists found that the toxicity that zinc oxide nanoparticles had for freshwater algae, bacteria and crustaceans resulted mainly from the organisms' exposure to dissolved zinc.
Similarly, dissolved silver was determined to be the cause of interrupted photosynthesis in freshwater algae exposed to silver nanoparticles. And in a study in South Korea, both silver nanoparticles and dissolved silver were shown to impede the reproductive potential of the nematode C. elegans, but the silver nanoparticles had a more potent effect and were observed to localize near the uterus.
Similarly, silver nanoparticles inhibited the functions of nitrifying bacteria more than dissolved silver did. And finally, one other study found that nanoparticulate iron was fatal to E. coli bacteria while dissolved forms of iron were not.
Still, in some cases the nanoparticles themselves have been shown to be toxic. A group of British researchers, for instance, observed that carbon nanotubes, among the most common form of ENPs, caused lung lesions in rats more quickly than asbestos did.
Too Early or Too Late
Although these findings share a common thread — identifying enhanced toxicity in the nanoparticle form of the substances under study — Kulinowski cautions that it is too early to make any generalized statements beyond saying that "some nanoparticles behave this way in some circumstances. We're all still struggling to understand precise mechanisms by which nanoparticles may cause harm to living organisms."
Still, she says, given the rapid growth of the industry and the many remaining questions concerning nanoparticle toxicology, "It is a legitimate concern as to whether potentially dangerous nanoparticles are getting into the environment while scientists try to figure out the mechanisms of any toxicological effects they may have."
Scientists may lack answers to many finer points of nanoparticle toxicity, but they do know that the environmental concerns derived from the same characteristics that may make nanoparticles instrumental in breakthrough technologies.
For instance, ENPs' extremely high ratio of surface area to volume significantly enhances their ability to bond to and react with other materials. Further, a substance that is insoluble as bulk material may become soluble as an ENP, enabling it to pass through a membrane, and non-conductive materials may become conductive in their nanoparticulate form.
Also, nanoparticles are so small that they can be inhaled easily, passing into the bloodstream and accumulating in organs. Healthy skin is thought to prevent the particles from entering the body, but the findings are not conclusive and, according to Holden, are the result of experiments conducted on skin that is not being stretched (as it is when a body or limb is bent or twisted). Some nanoparticles have shown a disturbing ability to pass through the olfactory nerve into the brain, evading the blood-brain barrier.
Despite all these characteristics, nanoparticles remain unregulated, and products incorporating them — currently numbering more than a thousand, according to the Project on Emerging Nanotechnologies — do not have to undergo any special testing for safety before being introduced into the marketplace.
"Regulation always lags behind commercialization, and that's true here. There are no nanospecific regulations on the books," says Kulinowski, adding, "The FDA doesn't regulate particles, it regulates products."
She explains that the Consumer Product Safety Commission has no pre-market approval required for nano-enabled products. The U.S. Environmental Protection Agency had a voluntary reporting program in which companies were invited to come forward with nano-enabled products and tell how they're used, but, says Kulinowski, "Not many signed up, so now they're going back to maybe being more formal with this."
Meanwhile, the International Organization for Standardization is working to produce a set of nanoparticle standards.
The search for answers is further complicated because the field of nanotoxicology is so new that standards, benchmarks and research protocols have not yet been established.
"We have lots of papers but it's difficult to tease out generalizable principles," Kulinowski says. One reason is that "protocols are different for different research projects. People are actually doing things like sending nanoparticles around the world and then having the same test conducted to see if perhaps the water is different in Japan and that is what's causing things to turn out differently there than in California. To try to establish protocols, we need a lot more of that kind of work."
Furthermore, she says, much of the early nanotoxicology work "will have to be re-evaluated and reported with better characterization," such as whether particles being tested were crystalline or amorphous, what medium they were tested in and whether they are coated or not.
And some of the groundwork laid so far is unstable. For instance, Kulinowski explains, researchers found that carbon nanotubes interact with dye molecules used in marking and "totally mess up the results, generating false positives or negatives."
There are also issues with measuring the dose that organisms are receiving in experiments.
"When you're dealing with chemicals as toxicants, you measure molarity, or chemical concentration," she says. "But there's no 'right' way to go about it for nanoparticles. Should we measure particle size, number of surfaces on a particle, surface area, or what's on the surface, which may be coated, or cloaked, with other materials? There is no consensus on what the measurements of dose should be."
What there is, however, is agreement that the race to understand nanotoxicology is on — and important.
"The establishment of principles and test procedures to ensure safe manufacture and use of nanomaterials in the marketplace is urgently required and achievable," Andre Nel and three co-authors wrote in 2006 in the journal Science. Nel is chief of the Division of NanoMedicine at the California NanoSystems Institute, director of Medicine at the David Geffen School of Medicine at UCLA, and director of UC CEIN.
"The problem right now is that we don't have the data to know, say, how much titanium dioxide is getting into a river bed and the effect it is having on aquatic life," says Kulinowski. "What we have is knowledge that it shouldn't get into the water because it can lead to bad outcomes. We need source data; we need to understand use and potential exposure in environments. We need to be able to establish acceptable levels of exposure."
"There is not yet a paradigm," says UCSB's Holden, who uses the accumulated body of knowledge on the toxicology of oil products as a parallel. "Oil is essentially a soup of all kinds of materials and compounds, but we have identified and studied the characteristics and toxicity of the various petroleum constituents enough to be able to make some general statements," she says. "We don't have this knowledge for nanoparticles."
Given the challenges to writing the complete book on nanotoxicology, Kulinowski finds hope in the rapidly growing awareness and broadening scientific discussion of the subject.
"We have come an enormous way from 2000 and 2001, the bad old days when people would say, 'Oh, we can't talk about that,'" she says. "Now we have whole centers focused on the risk side. The conversation is mainstream. There are a lot of very smart people working on this now, and we can articulate the questions so that scientists know what they want to find out when they go into the lab.
"The new mantra is: 'Safety by design,'" she continues. "So you figure out how to neutralize the toxicity of a nanoparticle before it gets into the water-treatment plant. You find out what the toxicological elements of a nanoparticle are and engineer those out."
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