Such rationality has been given a boost by the US Department of Energy's Brookhaven National Laboratory (Upton, NY), where scientists have developed a cell-culture screening method to examine how newly made nanoparticles interact with human cells. Their method is described in a review article about carbon-nanoparticle toxicity in a special section of Journal of Physics: Condensed Matter devoted to nanoscience and nanotechnology (18 S2185).

"Nanomaterials show great promise, but because of their extremely small size and unique properties, little is known about their effects on living systems," said lead author Barbara Panessa-Warren, a Brookhaven biologist who has been developing a nanoparticle cytotoxicity-screening model for the past five years. "Our experiments may provide scientists with information to help redesign nanoparticles to minimize safety concerns, and to optimize their use in health-related applications. They may also lead to effective screening practices for carbon-based materials."

Clinical promise

In the biomedical arena, nanoparticles are the subject of fast-moving development efforts in areas like drug delivery and diagnostic imaging (see "Special report: magnetic nanoparticles" and "Nanoparticles enhance ultrasound signals" on medicalphysicsweb). How those nanoparticles react with different types of living cell, hormone or immune factor is fundamental to the long-term clinical and commercial viability of such work. More significant is how nanoparticles react following biodegradation within the body - and specifically, whether the particles (or their by-products) are subject to bioaccumulation within cells or organs, inducing intracellular changes or inflammatory responses.

To date, numerous studies conducted in living animals have found a range of toxic effects resulting from exposure to carbon-based nanoparticles. All of these in vivo studies clearly show that multiple factors interact following nanoparticle exposure to produce acute and chronic changes within individual cells and the organism itself. In vitro laboratory studies, such as the cell-culture method developed by the Brookhaven team, are an attempt to simplify this field of research by eliminating many of the variables found in animal studies.

"By combining techniques of molecular biology with sophisticated imaging methods, we can rapidly gather information about the response of specific cell types to specific nanoparticles, making in vitro testing an inexpensive and immediate tool for screening and fine-tuning nanoparticle design to maximize safety and target specificity," explained Panessa-Warren.

In their studies, the Brookhaven scientists used lung and colon epithelial cells - chosen to represent likely routes of exposure via inhalation and ingestion - grown as cell monolayers (i.e. where the individual cells join together to form a tight layer with many of the characteristics of lung and colon cells growing in the body). These monolayers of living cells were subsequently exposed to varying doses of carbon nanoparticles over differing amounts of time, and the cells evaluated at each time period and dose.

Cellular responses

The researchers tested the response of the cells to different types of nanoparticle. They also assessed cell viability (whether the cells live or die) and growth characteristics of the monolayer, and examined any alterations in the cells using various microscopy techniques. As a consequence, they were able to visualize the first contact of the nanoparticles with the cells and follow this process "ultrastructurally". This meant they could see how the cells responded and determine whether the nanoparticles entered the cells or caused specific changes to the cell surfaces of those cells that did not die.

Panessa-Warren and colleagues found that a type of engineered carbon nanoparticle called a "nanoloop", which was made at Brookhaven, did not appear to be toxic to either cell type regardless of dose and time. In contrast, both colon and lung cells exposed to carbon nanoparticles from a raw nanotube preparation showed increased cell death with increased exposure time and dose. Microscopic studies revealed losses of cell-to-cell attachments in the monolayers, and changes in cell-surface morphology on cells where carbon nanotubes and other carbon nanoparticles had attached. Damage was severe for both the low and higher doses at 3 h, suggesting that exposure time may be even more predictive of damage than nanoparticle concentration.

Electron microscopy revealed that where the carbon nanoparticles, and especially carbon nanotubes, touched or attached to the cell surface, the plasma membranes became damaged and microscopically interrupted. Images of the cell surfaces with attached carbon nanoparticles showed membrane holes that exposed the underlying cell cytoplasm. What's more, transmission electron microscopy revealed that small carbon particles could pass into the cells and become incorporated into the cell nuclei. Neighbouring cells with no attached carbon nanoparticles appeared normal and continued to grow, suggesting that direct contact with untreated nanoparticles is required for damage to occur.

"Although our screening method gives us a quick way to analyse human cell responses to nanoparticles at a visual macro- and microscale, we are now taking this to a molecular and genetic level to see whether the cells are stressed," noted Panessa-Warren. Ultimately, though, any nanomaterials intended for large-scale production or use would also have to be tested in vivo - where the combined reactions of many cell types and tissues, as well as the blood, immune and hormonal factors, are all taken into account to assess biocompatibility and assure safety. "Still, our methods give us a way to screen out those nanoparticles that shouldn't even make it that far, or identify ways to improve them first," she added.

Take it as read, Brookhaven plans to be at the forefront of the international evidence-gathering on nanoparticle biocompatibility and toxicity. With this in mind, the laboratory is currently building a new centre for functional nanomaterials, with a five-year budget of $81 m that will enable it to focus on atomic-level tailoring of nanomaterials and nanoparticles across a range of scientific and biomedical disciplines.