Magnetic microdiscs target and initiate cell death in tumors

By ANN WANG
Issue date: 12/3/09

Scientists working at Argonne National Laboratory in Chicago and The University of Chicago have developed an effective method to target and kill cancer cells using tiny magnetic discs.

The microdiscs, only one micron in diameter, work by disrupting the outer membranes and initiating chemical pathways that lead to apoptosis, or cell death. In laboratory tests, the microdiscs destroyed up to 90 percent of cancer cells after being activated for only 10 minutes.

One major drawback of chemotherapy drugs, widely used to treat cancers, is that they cannot be targeted to tumor cells. These drugs affect the entire body and often cause painful side effects such as hair loss, nausea, fatigue and a weakened immune system.

For several decades, scientists have been trying to develop nanoparticles that can deliver drugs specifically to cancer cells. Although several such methods are now being tested in clinical trials, practical hurdles still remain.

Up until now, effective treatments required high concentrations of magnetic particles and high levels of power to activate them. Both could cause harmful side effects in patients.

The new research offers a potential solution to many of these problems. The team studied an aggressive brain cancer called glioblastoma multiforme. The surfaces of these cancer cells, called glioma cells, contain a much higher concentration of a protein called IL13 than normal cells do.

The microdiscs, each 60 nm wide and 1000 nm in diameter, were made of an iron and nickel alloy, then coated with a thin gold veneer. Gold is both nontoxic to living tissues and easy to modify with organic molecules. The gold-covered microdiscs were then coated with antibodies that would recognize and bind to the overexpressed protein on glioma cells.

Once introduced into the body, or in this case a cell culture, the antibodies guide the microdiscs to attach to the surface of the cancerous glioma cells, but not healthy cells. About 10 microdiscs attached to each cancer cell.

Because they are discs instead of particles, and much wider than they are thick, the microdiscs have a magnetic property known as a spin-vortex ground state, and they oscillate when an alternating current is applied.

The cell membrane consists of a fluid double layer of lipid molecules, more like the film that forms over a bowl of cold soup. This fluid membrane is easily disrupted by the twisting and turning motion of the microdiscs attached to its surface.

"The spin-vortex-mediated stimulus creates two dramatic effects: compromised integrity of the cellular membrane . . . and initiation of programmed cell death," said Elena Rozhkova, a research scientist at Argonne National Laboratory who worked on the study.

After they activated the microdiscs, the researchers noticed that most of the cancer cells looked like they were undergoing apoptosis, the controlled pathway towards death that normal cells are programmed to follow once they reach the end of their useful lives.

Cancer cells have developed mutations that allow them to escape cell cycle control and apoptosis. Instead of dying when they should, they divide and grow continuously, forming tumors.

However, the microdisc-treated glioma cells had fragmented DNA and nuclei, rounded shapes and irregular surface bulges (scientific term: blebs), all classic signs of cells undergoing apoptosis.

But the force the microdiscs exerted could not have caused such striking changes in the cells alone. In fact, the torque exerted by the microdiscs was less than one tenth of the torque needed even to break the outer membrane.

It was clear that the surface disruptions that the discs created were activating a signaling pathway inside the cell that led to apoptosis.

The researchers found that microdisc-treated cells had much higher concentrations of calcium than usual. Calcium plays a major role in many cell pathways and is known to be a key signaling molecule in apoptotic pathways.

Previous studies had also shown that even minor, temporary cell membrane disturbances can raise calcium levels within cells. It seems likely that the mechanical stimulus provided by the microdiscs is then amplified as a chemical signal inside the cell, leading the cell to begin apoptosis.

One key advancement in the team's research was that because of the material used to make the discs, a relatively low frequency and short treatment time was enough to kill most cancer cells. The relative mildness of the treatment may help decrease side effects in vivo.

"Using the unique 'soft' magnetic material allows application of a low-frequency field of a few tens of hertz applied for only ten minutes, [which] was sufficient to achieve approximately 90% cancer-cell destruction in vitro," Rozhkova said. "This is 10-100,000 times weaker magnetic field that it is used for superparamagnetic particles."

Although these new findings offer a promising new way that nanotechnology can be used to treat cancer, more work needs to be done before clinical trials can begin.

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Ref:

Ferromagnetic microdisks as carriers for biomedical applications

J. Appl. Phys. 105, 07B306 (2009); doi:10.1063/1.3061685

Published 5 March 2009

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ABSTRACT

REFERENCES (10)

CITING ARTICLES

E. A. Rozhkova,¹ V. Novosad,² D.-H. Kim,² J. Pearson,² R. Divan,¹ T. Rajh,¹ and S. D. Bader^2
1Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439, USA
²Materials Sciences Division, Argonne National Laboratory, Argonne, Illinois 60439, USA

We report the fabrication process, magnetic behavior, as well as the surface modification of ferromagnetic microdisks suspended in aqueous solution. They posses unique properties such as high magnetization of saturation, zero remanence due to spin vortex formation, intrinsic spin resonance at low frequencies, and the capability of delivering various biomolecules at once. Furthermore, because of their anisotropic shape, our magnetic particles rotate under alternating magnetic fields of small amplitude. This can be used to promote the idea of advanced therapies, which include combined drug delivery and magnetomechanical cell destruction when targeting tumor cells. The approach enables us to fabricate suitable magnetic carriers with excellent size tolerances, and then release them from the wafer into solution, ready for surface modification and therapeutic use. The particles have a magnetic core and are covered with few nanometers of gold on each side to provide stability at ambient conditions as well as biocompatibility and selective adhesion functions. A successful attempt to bind thiolates, including SH-modified antibody, to the disk's surface was demonstrated. ©2009 American Institute of Physics

History:	Presented 12 November 2008; received 13 October 2008; accepted 23 October 2008; published 5 March 2009
Permalink:	http://link.aip.org/link/?JAPIAU/105/07B306/1

New Target Against Flu Virus May Extend Vaccine Potency

Antibody Uncovers Vulnerability of Protein Stem

HMS researchers have found an Achilles heel in the influenza virus that may someday make annual flu shots a thing of the past. By targeting a hidden pocket in the microbe with a newly discovered antibody, they disabled a wide range of viruses, including those that cause the avian flu and the virulent 1918 Spanish flu.

While this research could lead to clinical trials of a new antiviral as soon as 2012 and may eventually lead to a more durable influenza vaccine, its influence may extend even further. The work, described in the March Nature Structural and Molecular Biology, validates a novel approach to finding such viral vulnerabilities and reveals what may be a more general principle for defeating a variety of pathogens.

Photo by Graham Ramsay

Wayne Marasco and Jianhua Sui have discovered influenza’s Achilles heel and devised a method to attack it.

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Striking Gold
The story begins in the lab of Wayne Marasco, HMS associate professor of medicine at the Dana–Farber Cancer Institute. Twelve years ago, Marasco collected blood from 57 healthy Bostonians and used the samples to create a library of 27 billion different human antibodies.

Researchers “pan” the library by presenting it with an antigen, such as a whole virus or a protein on the viral surface. Panning unearths antibodies that bind to that antigen. Marasco used his library to isolate an antibody against SARS in 2004.
When the avian flu appeared, Marasco and first author Jianhua Sui put the library to work again. But instead of panning with the whole H5N1 influenza virus, they focused on a single protein. They isolated the H5 version of hemagglutinin, a surface protein on influenza that allows the virus to invade a cell and replicate. (The N portion is a different surface protein called neuraminidase, which allows the virus to exit the cell.) The effort uncovered 10 potential antibodies.

Sui and Marasco, in partnership with co-author Rubin Donis, chief of the molecular virology and vaccines branch of the Centers for Disease Control and Prevention, tested three of these antibodies in mice infected with a lethal dose of avian flu. The antibodies neutralized between 80 and 100 percent of the infections.

Unexpectedly, the antibodies also neutralized other strains. They knocked down H1N1 (the 1918 Spanish flu), H2N2, H6N1 and more. “It became apparent very quickly that the target they were recognizing was highly conserved,” said Marasco.

Not only were these antibodies more broadly effective than expected, they also worked differently. Most antibodies stick to the round top of the lollipop-shaped hemagglutinin protein and interfere with the protein’s ability to bind to the cell membrane. But Marasco and Sui’s antibodies were not blocking the membrane binding. “That told us right there that the antibody wasn’t working against the globular head,” said Marasco.

Stemming the flu. Each year, scientists develop new influenza vaccines to target the ever-mutating globular heads (light red) of the hemagglutinin proteins that coat the virus. A newly discovered antibody binds to the much less variable and much less accessible stem of the protein (red). Work is under way to turn this antibody into an antiviral that can be used to contain a pandemic and to protect immunosuppressed individuals during flu season. Since the machinery of the stem evolves more slowly than the head, the discovery may lead to a broadly effective influenza vaccine that lasts for longer than a single season.

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At this point, a third part of the team became critically important. Robert Liddington and his team at the Burnham Institute for Medical Research crystallized one of the antibodies bound together with hemagglutinin. “That’s when the revelations started coming,” said Marasco.

The crystal confirmed that the antibody was not bound to the enticing top of the protein, but rather to a pocket in the stem. That pocket contains complex machinery. It houses three entangled moving parts that allow the virus to infect the cell (see video). The crystal revealed that the antibody grabs onto all three and prevents that machinery from working. “In the past, people didn’t even know to look in that pocket,” said Sui.

Sui took this information and used it to search a database of more than 6,000 (and growing) known genetic variants of the flu. She found that only two versions of this complex stem-based machinery have evolved. An examination by Liddington’s team of crystal structures of known variants found the same. The antibodies Sui and Marasco found work against one version. They are now running the other version of the stem through the same panning process to find an antibody against it.

Enduring Weakness
The contrast between the slow evolution of the stem and the impossible-to-keep-up-with evolution of the head is stunning. But it is not surprising. The part of the headpiece that binds to the cell membrane is very small, said Sui. So the rest of the headpiece can change dramatically without compromising the function.

But in the stem, “the delicate and complex machinery is highly conserved,” said Donis. “The virus cannot mutate it because by doing so, it would commit suicide.” Indeed, Donis’s team attempted to force mutations in the stem, but none emerged.

In discovering this new, hidden vulnerability, the researchers have realized that the virus has been fooling them, and our bodies, all along. “The virus has very cleverly developed an area on the top of its coat protein that creates a molecular decoy,” said Marasco. He speculates that the immune system mounts a full-scale attack against the easy-to-spot decoy while it simultaneously suppresses any efforts to target the elusive stem. Similarly, new vaccines chase the decoy each season hoping to hit it just right.

DOUBLE CLICK PIC FOR VIDEO

Courtesy Dana–Farber Cancer Institute

HMS researchers are targeting a common weakness to tackle influenza.

Courtesy Dana–Farber Cancer Institute

HMS researchers are targeting a common weakness to tackle influenza.

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But now, with the new insights from this work, “a pan-therapy for all kinds of influenza may be within our grasp,” said Liddington. Further, Marasco suspects that the influenza virus’s means of protecting its most vulnerable machinery may be a more general strategy. He has observed almost the exact same system in corona viruses, such as SARS.

Assuming that approval for human testing proceeds without a hitch, the new influenza antibody will likely be used as an antiviral first. Since it is unlikely that a mutant will evolve to defeat it, the hope is that this antiviral can be stockpiled and stored for years. Marasco also speculates that it may be possible to develop a vaccine that both masks the decoy and allows the immune system to attack the less flexible stem.

In the future, Marasco plans to apply these same methods to other viruses. The team’s approach not only allows them to find novel antibodies and hidden targets, it also helps researchers respond nimbly as resistant strains evolve.

—Elizabeth Dougherty

Students may contact Wayne Marasco at wayne_marasco@dfci.harvard.edu for more information.

Conflict Disclosure: The authors report no conflicts of interest.

Funding Sources: National Institutes of Health

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Immune cells show long-term memory

By Tina Hesman Saey

Web edition : Sunday, August 17th, 2008

Almost a century after exposure to the 1918 Spanish flu, survivors’ white blood cells still recognize the virus

Even after 90 years, the immune system doesn’t forget the face of a mass-murderer. A new study shows that survivors of the 1918 Spanish flu pandemic still have immune cells that remember the culprit virus.

Such long-lived immunity was thought to be impossible without periodic exposure to the microbe that stimulated the immune system in the first place. But a study published in advance online August 17 and slated for an upcoming issue of Nature reveals that immunity to a virus can last nearly a century.

“This is a really extraordinary finding,” says Peter Palese, a virologist at Mount Sinai School of Medicine in New York City who was not involved in the study. “It’s like immunological archaeology.”

Previous research showed that elderly people have antibodies that can recognize the 1918 flu virus, but that those antibodies usually also latch on to more recent viruses of the same subtype as the Spanish flu. The new study demonstrates that the immune system retains a specific memory only for the 1918 virus, which killed more than 20 million people worldwide.

Researchers led by viral immunologist James Crowe of Vanderbilt Medical Center in Nashville, Tenn., found a type of immune memory cell called B cells in the blood of elderly people who had lived through the 1918 Spanish flu pandemic. B cells are white blood cells that make antibodies against specific features of the proteins of an invading microbe.

In the pandemic survivors, about one in every 4.6 million B cells made antibodies that attack the 1918 virus but don’t latch on to more recent flu viruses that resemble the Spanish flu. That results offer evidence that the immune system remembers a virus for decades without being stimulated by reexposure, Palese says.

Although the 1918 pandemic was particularly virulent, the new study suggests the immune system can probably sustain a lifetime’s worth of defense against less deadly diseases as well, Palese says. And good vaccines should produce similar longevity in the immune response, possibly eliminating the need for frequent booster shots, he says.

Antibodies produced by the pandemic survivors are some of the most potent antibodies ever described, says Crowe. Mice given the antibodies and also infected with the 1918 virus survived.

“This is entirely counter to everyone’s intuition — that elderly people would have such potent antibodies,” Crowe says. Aging typically reduces a person’s ability to build antibodies and develop immunity to diseases, so it was a surprise to find that the elderly survivors of the Spanish flu could still mount such a vigorous defense against the virus.

Should the 1918 virus reappear, antibodies from the survivors might be used as a therapy to treat infected people, Crowe suggests. He and his colleagues produce the antibodies from cell cultures of the survivors’ B cells to prevent the need to keep drawing blood from the survivors.

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