20 JOHNS HOPKINS ENGINEER SUMMER 2006

LAB NOTES

Tiny Cubes May Hold Huge Potential

David H. Gracias fabricates metal microcontainers that one day could deliver drug therapies or relay health information from inside the body.

MEDICATIONS CAN ENTER the human body in many ways. In liquid form, they can be injected into the bloodstream. As a tablet or capsule, they can be swallowed. In tiny particle form, they can be inhaled into the nose.

Yet another type of medicine mover is on the horizon. David H. Gracias wants to put therapeutic drugs or helpful cells inside a box. A metal box, no larger than a dust speck. A self-assembling box constructed from a pattern of flat squares. A box that can be tracked by physicians who want to make sure it’s going where it will do the most good. A box that might even be controlled by a radio transmitter outside the body, allowing it to release its treat-ment precisely when and where it’s needed. A box that’s relatively inexpensive to mass-produce.

The project may sound like science fiction, but Gracias, who is an assistant professor in the Whiting School of Engineering’s Department of Chemical and Biomolecular Engineering, is off to a promising start. Last December, his research team published a paper in the journal Biomedical Microdevices, detailing how team members had constructed the tiny cubes and how they had, in a lab, successfully dispensed some microbeads and live cells com-monly used in medical treatment. The researchers also reported that they could locate the cubes inside a closed tube using magnetic resonance imaging (MRI), a common hospital technology.

The journal article stirred international attention for Gracias, who fabricates the microcubes in his laboratory on the Homewood campus. The article also represented the fruit of his collaboration with the Johns Hopkins School of Medicine’s In Vivo Cellular and Molecular Imaging Center, where post-doctoral fellow Barjor Gimi (lead author on the paper) and colleagues tracked the tiny metal boxes with MRI equipment. The paper generated excitement among researchers elsewhere at Hopkins and at other universities, including one team that believes the microcubes might provide a new way to deliver neurotransmitters to the brain.

Gracias’ project also caught the eye of a few venture capitalists who said they may consider funding further research and develop-ment in the technology if it continues to advance.

In March, the Arnold and Mabel Beckman Foundation award-ed Gracias a three-year grant totaling $264,000 to support his research on microcubes. As a 2006 Beckman Young Investigator, he will present at an August symposium in Irvine, California, at the Arnold and Mabel Beckman Center of the National Academies.

If the microcubes, perhaps equipped with tiny electronic com-ponents, prove to be a safe and effective new way to deliver therapy

into the body, Gracias envisions important real-world medical applications. He imagines diabetics who could signal microcubes in their body to release drugs to control blood sugar. Perhaps, he suggests, a person with epilepsy could direct the cubes to release crucial medication at the first hint of a seizure. Although he is not a physician, Gracias wonders if the microcubes might someday be used to deliver remedies directly to cancer cells or to a dangerous blood clot.

“We’re talking about an entirely new encapsulation and deliv-ery device that could lead to a new generation of ‘smart pills,’ ” Gracias says. “The long-term goal is to be able to implant a collec-tion of these therapeutic containers directly at the site of an injury or an illness. What we’ve done so far is a good first step, but it’s early. We still have a lot of work to do.” In the months ahead, the researchers must demonstrate that the metal cubes will not harm living cells over an extended period. Then, they must successfully test the cubes in lab animals. Trials involving human subjects could begin after that.

Gracias, who was born in Bombay, India, earned his doctorate at the University of California at Berkeley. He began working on the self-assembling microcubes while a post-doctoral fellow at Harvard University in the laboratory of George M. Whitesides. Later, he worked as an engineer for the Intel Corporation, develop-ing new processes for fabricating microchips. In 2003, Gracias joined the Whiting School’s faculty to focus his research on con-structing micro- and nanosystems with medical applications.

The perforated microcubes (above) are self-assembled with techniques borrowed from microelectronics. In his lab (right), David H. Gracias, an assistant professor of Chemical and Biomolecular Engineering, dons the attire needed to manufacture them in a clean room.

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Precision Hinges That MeltTo produce the self-assembling microcubes, Gracias borrowed some of the same techniques used to make microelectronic circuits: thin film deposition, photolithography, and electrodepo-sition. With these methods, he and his colleagues make a flat pattern of six squares in a shape resembling a cross. Each square, made of copper or nickel, has small pores etched into it. Medica-tions or therapeutic cells eventually could be loaded and released from the cube through the pores.

Along the edges of the adjoining squares, the researchers apply metallic solder to form hinges. These hinges melt when the squares are heated briefly in a lab solution. High surface tension in the liquefied solder pulls each pair of adjoining squares together like a swinging door. The result is a perforated cube. Cooling the solution causes the solder to harden again, and the containers retain their box-like shape.

“To make sure it folds itself exactly into a cube, we have to engineer the hinges very precisely,” Gracias says. “The self-assem-bly technique allows us to make a large number of these micro-containers at the same time and at a relatively low cost.”

The hope is that these microcubes someday can be injected into targeted body tissue, not into the bloodstream. In theory, medical personnel could use magnetic forces to move the metal boxes to a particular location once the boxes are in the body. Gracias and his colleagues have not yet demonstrated that this type of magnetic manipulation in the body would work, but they remain confident. They have, however, built microcubes that can release their contents in response to a radio signal.

How Safe Are They?As their research proceeds, one early hurdle is determining whether the metallic microcubes pose any toxic threats to living tissue. During the fabrication process, each cube receives a thin coating of gold, an inert metal that generally poses no toxicity problems in humans. But the researchers need to make sure this protective coating will last and that the cubes will trigger no dan-gerous reactions within the body.

These initial toxicity tests are under way in Gracias’ Homewood lab. Animal testing is expected to follow at another location. In the early experiments involving MRI tracking, Gracias collaborated with radiology researchers at the Johns Hopkins School of Medicine. He hopes to partner with other researchers there to conduct the animal testing, which is beyond his area of expertise. The Whiting School’s close ties with the School of Medicine are important, he

emphasizes. “We’ll need their help for the in vivo testing and more MRI tracking,” he says.

Gracias also is working with the University’s technology transfer staff, which has filed for a provisional patent covering the self-assembling microcontainer technology.

Bio-beeps from the BodyAlthough drug delivery applications have received the most attention, Gracias believes the microcubes have other potential uses. If their ability to house radio components is developed, the cubes may be able to function as bio-sensors, relaying important health information from inside the body without the need for major surgery.

Another interesting prospect, Gracias says, is the placement of two different compounds in separate microcubes. When the cubes reach the right location, the contents could be released simultaneously and interact to create a chemical reaction in a tightly confined site. “This could have implications not just in medicine, where some drugs need to be mixed just before they are used, but also in chemistry,” he notes, referring to new nano-fluidic technology, in which liquid flows through tiny maze-like channels on a chip.

For Gracias, the future is wide open. “Our self-assembling cubes,” he says, “have generated a lot of excitement.”

— Phil Sneiderman

To learn more, visit engineering.jhu.edu/chbe/faculty/gracias/g/.

LAB NOTES

“The self-assembly technique allows us to make a large number of these microcontainers at the same time and at a relatively low cost.” — David H. Gracias

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22 JOHNS HOPKINS ENGINEER SUMMER 2006

Getting the Pollutant Priorities StraightIn their hunt for emerging contaminants in the water supply, A. Lynn Roberts and Edward J. Bouwer probe beyond the usual suspects.

WITH ABOUT 80,000 CHEMICALS being produced in the United States, even the most diligent environmental watchdogs have to prioritize the list of pollutants to be monitored and regu-lated. In the 1970s, when the U.S. Environmental Protection Agency (EPA) created a list of 129 “priority pollutants,” it was the result of an extensive screening process. What chemicals were most toxic? Which were most common? Which were most likely to concentrate? How do they move through the environment?

The criteria made sense, but there was one other deciding factor that must now be revisited, according to A. Lynn Roberts, professor in the Whiting School of Engineering’s Department of Geography and Environmental Engineering (DoGEE): “We should have recog-nized that advances in environmental chemistry tend to be driven by advances in analytical instrumentation.” Her point is that the EPA didn’t put a chemical on the list if scientists didn’t know how to measure it, and their capabilities 30 years ago were much more limited than they are today.

“The techniques for environmental analysis are very powerful now,” Roberts says, “with phenomenal sensitivity down to a fraction of a nanogram in a liter of water. But you have to key in on a specific compound. If you’re not looking for something, you won’t see it.”

That’s why a group headed by Roberts and fellow DoGEE pro-fessor Edward J. Bouwer is looking at what are known as “emerging contaminants.” These are substances that now can be detected and monitored and may deserve serious attention from the scientific and regulatory communities. With advanced instrumentation at their disposal, they are ferreting out those chemical pollutants that previ-ously slipped under the radar. As Bouwer says, “A lot of the easy questions have been answered. Now we’re looking at compounds that are more complex, more esoteric.”

In a project funded by the international Awwa Research Foundation, Michelle Hladik ’06 PhD, as a graduate student in the Roberts Lab research group, applied some “chemical intuition” to come up with 20 compounds she thought were likely to be present in the water supply. Testing water from facilities in the Midwest, she detected 19 of the 20 chemicals. Testing waters from the Chesapeake Bay, she again found 19 of the 20. Her study indicated that conventional water-treatment techniques are not efficient at removing these pollutants. In one instance, Hladik found that while a particular herbicide was successfully broken down by chlorina-

LAB NOTES

“A lot of the easy questions have been answered. Now we’re looking at compounds that are more complex, more esoteric.”

— Edward J. Bouwer

JOHNS HOPKINS ENGINEER SUMMER 2006 23

tion, the process created a degradation product that was potentially even more dangerous that the herbicide itself. Says Roberts about such emerging concerns, “We’re just transmuting the problem, lulling people into a false sense of security.”

Roberts and Bouwer also are investigating a range of other pos-sible sources of emerging pollutants that could not be measured in the 1970s, sometimes simply because they didn’t yet exist. They include pharmaceuticals, personal care products, and antiseptics. Some deserve attention because we used to think they were broken down in sewage treatment plants when, in fact, they are not. Others, we now realize, accumulate very quickly. Still others have become abundant as micropollutants because of the high volume of drugs consumed in this country.

With help from graduate students Kevin Bisceglia and Jim Yu, Roberts and Bouwer are conducting extensive studies of pharma-ceuticals. They are reviewing the top 200 prescribed drugs, the top 200 over-the-counter drugs, the top 200 generics, and so on. “We have now done sampling in some sewage treatment plants along the Northeast coast,” says Bouwer. “Many of the target pharmaceu-tical compounds are being removed partially but still are coming out in the effluents. We have also done parallel laboratory studies that mimic field conditions so we can do tests on a small scale at low cost and under controlled conditions.” By discovering the fate of these chemicals and publishing the results, Bouwer and Roberts will help determine if any of the compounds belong on the EPA’s Candidate Contaminant List.

In a society that constantly generates new substances that may pose environmental or health risks, these DoGEE researchers will have plenty to keep them busy. As Roberts says, “We just keep handing Pandora new boxes, and she just keeps opening them.”

—Bob Cooke

To learn more about the work of A. Lynn Roberts and Edward J. Bouwer, visit engineering.jhu.edu/~dogee.

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IRK (Left) Today, many more potential chemical

pollutants can be detected using advanced instrumentation. DoGEE colleagues A. Lynn Roberts and Edward J. Bouwer seek those that might have slipped under the radar. (Right) These 20 compounds are the ones that Michelle Hladik ’06 PhD, while a graduate student in the Roberts Lab research group, suspected might be found in the water supply. They are neutral degradates of the herbicides alachor, metolachlor, acetochlor, and dimethenamid. She found 19 of them in water samples in the Chesapeake Bay and the Midwest. Hladik is now a chemist with the U.S. Geological Survey in Sacramento.