Few things can make a scientist’s day like a phone call from the Defense Advanced Research Projects Agency, or DARPA. A once-obscure branch of the Pentagon, DARPA gained wider recognition in the late 1990s as a kind of cloak-and-dagger agency, seeking out scientists doing the most audacious, out-of-the-box science it could find and enlisting them in projects to ensure national security.
Liza Green, HMS Media Services
“Magnetism is always at the basis of any magic trick—it’s under the table or wherever,” said Donald Ingber. “Magnetism also has definite effects in the body.” Ingber (front) is shown with (clockwise from left) Flavia Cassiola, Martin Montoya-Zavala, and Robert Mannix.
In the aftermath of the Gulf War, it was especially concerned with finding ways to fend off the threat of biological weapons and was convinced that answers lay in the cellular world. Cells carry out a host of functions—they can sense changes in the environment, process information, mount responses, and transmit signals. Immune cells, in particular, can detect and immediately respond to pathogens. DARPA wanted to harness this power to create small, cell-based, wearable devices that might protect American soldiers, and even civilians, from harmful biological agents. But they needed a portable, low-energy toggle that would quickly turn the cells on and off. They became intrigued by the possibility of creating a magnetic switch. It was around this time, early 2002, that DARPA phoned Donald Ingber.
“They asked, ‘Do you think this is feasible and how would you go about doing it?’” said Ingber, the Judah Folkman professor of vascular biology in the Department of Pathology at Children’s Hospital Boston. Ingber would spend the next few months coming up with a blueprint for such a device. Over the past five years, funded by a DARPA grant, he, Robert Mannix, Sanjay Kumar, and colleagues have been working to turn it into a reality.
In the January Nature Nanotechnology, they report that they have created a nanomagnetic cellular switch—one that can rapidly and reliably activate mast cells, a class of immune cell. Turning on the switch resulted in the release of intracellular calcium, which is exactly what happens when the mast cells are activated naturally.
Even more remarkable is how the switch mimics true mast cell activation. Mast cells are immune workhorses. They detect an extraordinarily wide variety of antigens—from pathogens and environmental contaminants—and attack them by releasing calcium, leading to the export of histamines and other substances. Like many cells, mast cells are not activated one receptor at a time. Antigens bind not just to one but several receptors on the cell surface, essentially drawing them together into clusters, or scaffolds, onto which activating molecules can fit.
Ingber’s concept was to recreate this clustering by attaching a single tiny iron oxide bead to each mast cell receptor and exposing the cells to a magnetic field. Once exposed, the beads would become magnetized, attracting one another and, at the same time, pulling the receptors into scaffold-like clusters (see figures).
“The idea that the biochemistry is really about binding sites in spatial proximity, that it’s really the physicality of bringing them together—that was the gamble, the hypothesis.”
“The idea that the biochemistry is really about binding sites in spatial proximity, that it’s really the physicality of bringing them together—that was the gamble, the hypothesis. If we could do this with magnets, would that be enough to trigger activation?” Ingber said.
Though the answer appears to be yes, the switch is still very much in the prototype stage. “It’s early in the process, and I think it needs to be taken a bit further to really have a specific target,” he said. But receptor clustering is a very common process and, down the road, the nanomagnetic switch could open the door to an array of cell-based devices.
“Imagine you engineer a cell to produce an antidote to some chemical biological agent,” Ingber ventured. “You cou.ld inject these cells subcutaneously—they’re basically factories that don’t normally make the antidote. With these particles on their receptors and a detector that says the agents are coming, you can switch the cells on to produce the antidote.” He envisions this might be done with a local magnet, lodged perhaps in a wristwatch. “The whole point is magnetic fields travel across the skin,” he said.
Getting Physical
Ingber is known for this kind of exuberant thinking—and, indeed, it is not surprising that DARPA called on him. Over the past three decades, he has been a lone voice for a different, often contrarian way of looking at cells and molecules—one that combines cutting-edge technology with an almost old-fashioned reverence for physicality (see Focus Sept. 3, 1999, and Feb. 25, 2005).
Years ago, in a set of classic experiments initiated while working as a postdoc with the late Judah Folkman, Ingber found that by varying the degree to which cells were stretched on a patch of extracellular matrix, he could get them to either grow, differentiate, or divide. It was while working on these experiments that he made his first foray into the field of magnetism. To get cells to stretch, he plated them on a bed of tiny magnetic beads, which the cells engulfed, and then exposed the cells to a magnet.
“I put my magnet underneath and all the beads aligned vertically, like little actin polymers. And the cells just sat between them—I knew nothing about magnetics,” he said. The beads had turned into tiny magnets and aligned in north–south fashion. He realized that for the beads to actually pull on cells required exposing them to a differential force, or magnetic gradient. He would go on to use the gradient-exposed iron oxide beads to explore the effect of mechanical force on integrins and other cell surface structures.
Divvying the Beads
But at five microns, the beads were far too bulky for the DARPA challenge. Ingber’s concept was to have one bead per receptor, which meant they needed to be infinitesimally smaller, about 30 nanometers. Also, the beads were supposed to tug on one another, pulling the receptors with them. Ingber wanted them to turn into tiny magnets when exposed to a magnetic field. In fact, to act as true on-off switches, they should be capable of being repeatedly magnetized and demagnetized, which sent Mannix, an HMS research fellow in pathology at Children’s, on a search.
“I began looking like crazy on the internet for beads and found a little company which stopped making the beads soon after I purchased them,” he said.
Beads in hand, Mannix, Kumar, then a postdoc in Ingber’s lab, and Ingber, faced another thorny challenge—making sure that each mast cell receptor had only one bead. They solved this by placing a non-binding ligand on 29 of the bead’s 30 binding sites, leaving only one with an actively binding ligand. Each receptor had been bound to a complementary antibody. The researchers mixed the ligand-coated beads with the antibody-bound mast cells. “Statistically, you can expect only one bead per receptor,” Mannix said. In fact, follow-up with scanning electron microscopy (SEM) by Flavia Cassiola, HMS research fellow in surgery, showed that this was the case.
Beads of a feather. Like many cells, mast cells are not activated one receptor at a time, but instead by the clustering of receptors. Multivalent ligands bind to not just one but several receptors on the mast cell surface, essentially drawing them together into clusters, or scaffolds, onto which activating molecules can fit (top). The researchers mimicked this receptor clustering by attaching a single tiny iron oxide bead to each mast cell receptor and exposing the cells to a magnetic field. Once exposed, the beads became magnetized, attracting one another and, at the same time, pulling the receptors into scaffoldlike clusters (middle). Scanning electron micrographs vividly show the mast cell receptors before and after clustering (bottom).
Mannix and Kumar, now an assistant professor of bioengineering at UC-Berkeley, then loaded the mast cells with a calcium-revealing fluorescent dye and placed them on a microscope stage. Placing the tip of a magnetic needle—crafted by HMS instructor in surgery Martin Montoya-Zavala—near the cells, they applied a magnetic field at regular time intervals. Analyzing photos taken during the experiment, the researchers could see that the nanomagnetic switches worked: when the magnetic field was applied, the cells fluoresced, indicating they had been activated. SEM of the mast cell surface showed that the fluorescing coincided with the clustering of receptors.
DARPA’s challenge was to develop a generic switch—one that could be used on a variety of cells. It remains to be seen whether the new switch works on other cells. But given that most cells employ receptor clustering, it should. Though their DARPA grant has run out, Ingber believes a follow-up project is an example of the kind of research that might be conducted under the auspices of the new Harvard Institute for Biologically Inspired Engineering, which he cofounded.
Meanwhile, Ingber and the Department of Defense (DoD) have forged a new and somewhat unexpected relationship. It turns out the DoD is one of the biggest funders of breast cancer research, and Ingber was recently granted one of their Breast Cancer Innovator Awards, suggesting his days as a maverick are far from over. But with the formation of a new institute, his is no longer a lone voice.
“In the past, bioengineers took engineering principles to solve biological problems, but the boundaries between living and nonliving are breaking down,” he said. “It’s really exciting.”
—Misia Landau
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