Why carbon nanotubes spell trouble for cells

It's been long known that asbestos spells trouble for human cells. Scientists have seen cells stabbed with spiky, long asbestos fibers, and the image is gory: Part of the fiber is protruding from the cell, like a quivering arrow that's found its mark.

But scientists had been unable to understand why cells would be interested in asbestos fibers and other materials at the nanoscale that are too long to be fully ingested. Now a group of researchers at Brown University explains what happens. Through molecular simulations and experiments, the team reports in Nature Nanotechnology that certain nanomaterials, such as carbon nanotubes, enter cells tip-first and almost always at a 90-degree angle. The orientation ends up fooling the cell; by taking in the rounded tip first, the cell mistakes the particle for a sphere, rather than a long cylinder. By the time the cell realizes the material is too long to be fully ingested, it's too late.

"It's as if we would eat a lollipop that's longer than us," said Huajian Gao, professor of engineering at Brown and the paper's corresponding author. "It would get stuck."

The research is important because nanomaterials like carbon nanotubes have promise in medicine, such as acting as vehicles to transport drugs to specific cells or to specific locations in the human body. If scientists can fully understand how nanomaterials interact with cells, then they can conceivably design products that help cells rather than harm them.

"If we can fully understand (nanomaterial-cell dynamics), we can make other tubes that can control how cells interact with nanomaterials and not be toxic," Gao said. "We ultimately want to stop the attraction between the nanotip and the cell."

Like asbestos fibers, commercially available carbon nanotubes and gold nanowires have rounded tips that often range from 10 to 100 nanometers in diameter. Size is important here; the diameter fits well within the cell's parameters for what it can handle. Brushing up against the nanotube, special proteins called receptors on the cell spring into action, clustering and bending the membrane wall to wrap the cell around the nanotube tip in a sequence that the authors call "tip recognition." As this occurs, the nanotube is tipped to a 90-degree angle, which reduces the amount of energy needed for the cell to engulf the particle.

Once the engulfing — endocytosis — begins, there is no turning back. Within minutes, the cell senses it can't fully engulf the nanostructure and essentially dials 911. "At this stage, it's too late," Gao said. "It's in trouble and calls for help, triggering an immune response that can cause repeated inflammation."

The team hypothesized the interaction using coarse-grained molecular dynamic simulations and capped multiwalled carbon nanotubes. In experiments involving nanotubes and gold nanowires and mouse liver cells and human mesothelial cells, the nanomaterials entered the cells tip-first and at a 90-degree angle about 90 percent of the time, the researchers report.

"We thought the tube was going to lie on the cell membrane to obtain more binding sites. However, our simulations revealed the tube steadily rotating to a high-entry degree, with its tip being fully wrapped," said Xinghua Shi, first author on the paper who earned his doctorate at Brown and is at the Chinese Academy of Sciences in Beijing. "It is counter-intuitive and is mainly due to the bending energy release as the membrane is wrapping the tube."

The team would like to study whether nanotubes without rounded tips — or less rigid nanomaterials such as nanoribbons — pose the same dilemma for cells.

"Interestingly, if the rounded tip of a carbon nanotube is cut off (meaning the tube is open and hollow), the tube lies on the cell membrane, instead of entering the cell at a high-degree-angle," Shi said.

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Injectable gel could spell relief for arthritis sufferers

Some 25 million people in the United States alone suffer from rheumatoid arthritis or its cousin osteoarthritis, diseases characterized by often debilitating pain in the joints. Now researchers at Brigham and Women's Hospital (BWH) report an injectable gel that could spell the future for treating these diseases and others.

Among its advantages, the gel could allow the targeted release of medicine at an affected joint, and could dispense that medicine on demand in response to enzymes associated with arthritic flare-ups.

"We think that this platform could be useful for multiple medical applications including the localized treatment of cancer, ocular disease, and cardiovascular disease," said Jeffrey Karp, leader of the research and co-director of the Center for Regenerative Therapeutics at BWH.

Karp will present the findings April 15 at the annual meeting of the Society for Biomaterials (SFB) as part of winning the coveted SFB Young Investigator Award for this work. The work was also reported by Karp and colleagues in the May 2011 issue of the Journal of Biomedical Materials Research (JBMR): Part A, and is currently available on the journal's website.

Local Delivery

Arthritis is a good example of a disease that attacks specific parts of the body. Conventional treatments for it, however, largely involve drugs taken orally. Not only do these take a while (often weeks) to exert their effects, they can have additional side effects. That is because the drug is dispersed throughout the body, not just at the affected joint. Further, high concentrations of the drug are necessary to deliver enough to the affected joint, which runs the risk of toxicity.

"There are many instances where we would like to deliver drugs to a specific location, but it's very challenging to do so without encountering major barriers," says Karp, who also holds appointments through Harvard Medical School (HMS), Harvard Stem Cell Institute (HSCI), and the Harvard-MIT Division of Health Sciences and Technology (HST).

For example, you could inject a drug into the target area, but it won't last long--only minutes to hours--because it is removed by the body's highly efficient lymphatic system. What about implantable drug-delivery devices? Most of these are composed of stiff materials that in a dynamic environment like a joint can rub and cause inflammation on their own. Further, most of these devices release medicine continuously--even when it's not needed. Arthritis, for example, occurs in cycles characterized by flare-ups then remission.

Toward the Holy Grail

"The Holy Grail of drug delivery is an autonomous system that [meters] the amount of drug released in response to a biological stimulus, ensuring that the drug is released only when needed at a therapeutically relevant concentration," Karp and colleagues write in JBMR. His coauthors are Praveen Kumar Vemula, Nathaniel Campbell, and Abdullah Syed of BWH, HMS and HSCI; Eric Boilard (now at Université Laval), Melaku Muluneh, and David Weitz of Harvard University; and David Lee of BWH, currently at Novartis. Karp notes the key involvement of Lee, a doctor who is "treating patients with the problem we're trying to solve."

The researchers tackled the problem by first determining the key criteria for a successful locally administered arthritis treatment. In addition to having the ability to release drug on demand, for example, the delivery vehicle should be injectable through a small needle and allow high concentrations of the drug. The team ultimately determined that an injectable gel seemed most promising.

Next step: what would the gel be made of? To cut the time involved in bringing a new technology to market, the team focused only on materials already designated by the Food and Drug Administration as being generally recognized as safe (GRAS) for use in humans.

Ultimately, they discovered a GRAS material that could be coaxed into self-assembling into a drug-containing gel. "The beauty of self-assembly is that whatever exists in solution during the assembly process--in this case, a drug--becomes entrapped," says Vemula, first author of the paper, who also has an appointment at HST.

They further expected that the same material would disassemble, releasing its drug payload, when exposed to the enzymes present during inflammations like those associated with arthritis.

Promising Results

A series of experiments confirmed this. For example, the team created a gel containing a dye as a stand-in for a drug, then exposed it to enzymes associated with arthritis. The drug was released. Further, the addition of agents that inhibited the enzymes stopped the release, indicating that the gel "can release encapsulated agents in an on-demand manner," the researchers write. Although the team has yet to test this in humans, they did find that dye was also released in response to synovial fluid taken from arthritic human joints.

Among other promising results, the researchers found that gel injected into the healthy joints of mice remained stable for at least two months. Further, the gel withstood wear and tear representative of conditions in a moving joint.

Additional tests in mice are underway. The technique has yet to be demonstrated in humans, but the researchers write that it "should have broad implications for the localized treatment of many…diseases" caused by the enzymatic destruction of tissues.

The researchers have applied for a patent on the work, which was sponsored by the Center for Integration of Medicine and Innovative Technology (CIMIT) through the U.S. Army and by the Harvard Catalyst Program.

Provided by Brigham and Women's Hospital