New methods for screening nanoparticles and interaction with human cells

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New methods for screening nanoparticles and interaction with human

cells

http://www.news-medical.net/?id=19579

Scientists at the U.S. Department of Energy's Brookhaven National

Laboratory have developed a screening method to examine how newly

made nanoparticles - particles with dimensions on the order of

billionths of a meter - interact with human cells following exposure

for various times and doses.

This has led to the visualization of how human cells interact with

some specific types of carbon nanoparticles. The method is described

in a review article on carbon nanoparticle toxicity in a special

section of the August 23, 2006, issue of the Journal of Physics:

Condensed Matter devoted to developments in nanoscience and

nanotechnology, now available online.

Nanoparticles may have different physical, chemical, electrical, and

optical properties than occur in bulk samples of the same material,

in part due to the increased surface area to volume ratio at the

nanoscale. Many scientists believe that understanding these nanoscale

properties and finding ways to engineer new nanomaterials will have

revolutionary impacts -- from more efficient energy generation and

data storage to improved methods for diagnosing and treating disease.

Brookhaven Lab is currently building a Center for Functional

Nanomaterials (CFN) with state-of-the-art facilities for the

fabrication and study of nanomaterials, with an emphasis on atomic-

level tailoring of nanomaterials and nanoparticles to achieve desired

properties and functions.

" 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. "

A variety of studies conducted in living animals, which are described

in the review article, 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 the research by eliminating many of

the variables found in animal studies, giving researchers greater

control over experimental conditions.

" 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, " Panessa-Warren said.

In the Brookhaven team's studies, the scientists used lung and colon

epithelial cells -- chosen to represent two likely routes of

nanoparticle exposure (inhalation and ingestion) -- grown as cell

monolayers, 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 as an epithelial layer. These monolayers of

living cells are then exposed to varying doses of carbon

nanoparticles over differing amounts of time, and the cells are

studied at each time period and dose.

The scientists also tested the response of the cells to different

types of nanoparticles (a raw nanotube preparation containing mostly

single-walled carbon nanotubes, nanoropes, graphene and trace

elements; partially cleaned air-oxidized carbon nanotubes; as well

as, carbon-nanotube-derived loops used to carry antibodies). They

assessed cell viability (did the cells live or die?) and growth

characteristics of the monolayer, and examined any alterations within

the cells using various microscopy techniques. These techniques

enabled them to visualize the first contact of the nanoparticles with

the cells and follow this process " ultrastructurally " so 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.

Using this in vitro screening, the scientists 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 the 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 three hours,

suggesting that exposure time may be even more predictive of damage

than nanoparticle concentration.

Using electron microscopy, the scientists found that areas in which

the carbon nanoparticles, and especially carbon nanotubes, touched or

attached to the cell surface, the plasma membranes became damaged and

were microscopically interrupted. Images of the cell surfaces with

attached carbon nanoparticles showed membrane holes that exposed the

underlying cell cytoplasm. Transmission electron microscopy revealed

that small carbon particles could pass into the cells and become

incorporated into the cell nuclei. Neighboring 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. These findings agree with recent

biochemical studies in the literature that reported the production of

reactive oxygen species (free radicals) and lipid peroxidation of

cell membranes following living cell contact with other forms of

carbon nanoparticles called fullerenes, also known as " buckyballs. "

" Although our screening method gives us a quick way to analyze human

cell responses to nanoparticles at a visual macro- and micro- scale,

we are now taking this to a molecular and genetic level to see

whether the cells are stressed, " said Pannessa-Warren.

" Ultimately any new 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, " she added. " 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. "

http://www.bnl.gov