Tuesday, May 10, 2011

An overlooked detail of experimental design may invalidate some prior experiments with nanoparticles

Nanoparticles will soon be used as tiny shuttles to deliver genes to cells and drugs to tumors in a more targeted way than was possible in the past.

But as the scientists prepare to use the nanoparticles in medicine, concerns have arisen about their potential toxicity.

Studies of both the applications of nanoparticles and their toxicity rely on the ability of scientists to quantify the interaction between the nanoparticles and cells, particularly the uptake (ingestion) of nanoparticles by cells.

In the standard laboratory tests of the biological activity of nanoparticles, cells are plated on the bottom of a dish and culture medium containing nanoparticles is poured on top of them.

It seems straightforward enough. But recently Washington University in St. Louis scientist Younan Xia started to worry about the in vitro experiments his lab was doing with gold nanoparticles.

What if the cells were upside down, he wondered? Would that make a difference? Would it change their uptake rate?

The swan‘s neck effect

As any bench scientist will tell you, it can be difficult to recognize, much less eliminate, the many extraneous factors that might bias an experiment.

One famous example is spontaneous generation — the idea that living organisms such as maggots arise from dead matter. Known to Aristotle, it persisted into the 19th century because people who tried to test it had trouble with experimental design.

They were particularly vexed by air. Should air be included or excluded from the flask holding a nourishing broth? Air might be necessary for spontaneous generation, as it is for combustion, or it might waft in microorganisms whose presence would render a positive result meaningingless.

So spontaneous generation wasn’t definitively disproved until the French scientist Louis Pasteur came up with an experimental design that separated the two roles of air. He boiled broth in a flask, then heated the neck of the flask and bent it into a swan’s neck.

Air could enter the flask, but microorgansisms in the air settled out in the bend in the neck.

The broth stayed clear, definitively proving that life does not arise spontaneously. Life comes only from life. PHOTO CREDIT: Yassine Mrabet/Wikimedia Commons
“People assumed that if they prepared a suspension, the suspension was going to have the same concentration everywhere, including at the surface of the cells,” says Xia, PhD, the James M. McKelvey Professor in the Department of Biomedical Engineering.

A battery of experiments in Xia’s lab with both the standard and upside-down setups showed that nanoparticles above certain sizes and weights will settle out. So concentrations of the nanoparticles near the cell surfaces are different from those in the bulk solution and cellular uptake rates are higher.

As Xia and his colleagues concluded in the Nature Nanotechnology article describing the experiments, “Studies on the cellular uptake of nanoparticles that have been conducted with cells in the upright configuration may have given rise to erroneous and misleading data.”

Topsies and turveys, Scientists have felt they could safely assume that the concentration of nanoparticles in the fluid next to the cells, which drives cellular uptake, was the same as the initial concentration of nanoparticles in the medium because the particles are small enough to be easily lofted by Brownian motion, the random motion of the molecules in the liquid.

Gravity, by this accounting, did not override this force for diffusion and the nanoparticles stayed in solution instead of settling out.

“We started to wonder, however, because our nanoparticles are made of gold,” Xia says. “Gold is nontoxic but it is also very heavy, so it was conceivable relatively large nanoparticles might settle.”

Because it is impossible to measure the exact concentration of gold nanoparticles at the surface of a cell, Xia and coworkers designed a simple experiment to test whether settling changed the concentration there and the cellular uptake.

Xia’s lab tested gold nanospheres of three sizes, nanocages of two edge lengths, and nanorods, some with surface coatings that picked up serum proteins in solution and others coated with a chemical that acts as an antifouling agent.

After the cells were incubated in the nanoparticle-bearing medium, the concentration of the nanoparticles in the medium was measured spectroscopically and the number of particles each cell had taken up was calculated from the difference in the concentrations.

In the literature, Xia says, there are reports that the cellular uptake of nanoparticles depends on the nanoparticles’ size, shape and surface coating.

His lab’s experiments showed that these characteristics are secondary, relevant only insofar as they affect the sedimentation and diffusion velocities of the nanoparticles.

For small, light particles, there was no disparity between the cells in the upright and the upside-down configurations. In the case of larger, heavier particles, however, sedimentation dominated, and the upright cells took in more nanoparticles than the upside-down cells.

“All earlier work may need to be re-evaluated to account for the effects of sedimentation on nanoparticle dosimetry,” the authors conclude.

“It’s no different from medicines that have to be shaken to suspend a powder in a water. If you don’t shake the bottle,” Xia says, “you end up under- or overdosing yourself.”

Contact: Diana Lutz dlutz@wustl.edu 314-935-5272 Washington University in St. Louis

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