Can nanomedicine end endotoxemia?

Jesse in the pasture, fal.A previous study conducted in the United States showed that eight out of 10 horses experience colic during their lifetime. Of those affected horses, 40 per cent of them die, likely as a result of endotoxemia – a complicating factor in many common equine diseases like colic and metritis (inflammation of the uterus).

Endotoxemia in horses occurs when the circulating blood is contaminated with endotoxin – a product released from bacterial cell walls when the cells die. And with no effective methods to treat and manage the disorder, endotoxemia continues to have devastating health and economic consequences within the horse industry.

Discovering a successful treatment for a complex disease such as endotoxemia is extremely difficult. However, Dr. Baljit Singh, a Western College of Veterinary Medicine (WCVM) professor and the College’s acting associate dean of research, enjoys the challenge and has been working with his research team for three years now to determine if nanomedicine is the key to treating this deadly condition.

The disease is highly significant for horses due to their extreme sensitivity to endotoxin exposure. “Horses are unique in that they possess a large number of macrophages (one type of an immune cell) in the capillaries of their lungs,” says Singh. “Many other species do not have these macrophages so I think this is the main reason horses tend to be more sensitive.”

When endotoxins come along in the blood, these macrophages are the first to pick them up, recognize them and produce inflammatory products in response to them.

Often it’s not the bacteria or the endotoxin that causes endotoxemic death: it’s actually the over-activation of the body’s immune system that kills the horse. “Large numbers of activated immune cells release necessary products for host defense, but in excessive amounts that cause damage to healthy tissue including blood vessels,” explains Singh. “This changes the blood flow to various organs resulting in low blood volume and eventually shock.”

Targeting neutrophils and macrophages, the two major cells of the immune system, and interfering with the inflammatory process are the top goals for Singh and his team.

“When neutrophils become activated, they not only release enzymes and reactive oxygen molecules in excess, but they also live longer – extending their life by almost 50 per cent,” says Singh. “We want to silence the neutrophils when they’re activated too much. When a disease process begins, we want to intervene with a signal telling the cells, ‘Enough. You’ve done your job. Now you can go home.’”

Tiny Solution to a Big Problem?
To tackle this immune problem, Singh is looking to nanotechnology for a solution. Nanotechnology is the ability to develop new materials by manipulating them on an extremely small scale. The nanometer scale (one billionth of a metre) is used in many everyday items including sunscreens, anti-aging creams and bandage materials.

“Particles or materials behave very differently based on their size,” says Singh. For example, silver-impregnated bandages used for wound management are only effective when the silver is broken down into small nanoparticles.

Not an entirely new concept for him, Singh began exploring the uses of nanomedicine in 2003 when a student, Shane Journeay, came to him for his PhD program.

“Shane wanted to do his PhD on nanoparticles,” says Singh. “As always, I’m very open to what students tell me rather than me telling them what to do. When he came along, I had to find out about the people involved in the area and what was current and new in that field. He essentially opened up and set up this new area in my lab.”

Through his research, Singh eventually came to know Dr. Hicham Fenniri, a University of Alberta professor and member of the National Institute for Nanotechnology (NINT). Fenniri designed the rosette nanotube – a novel nanomedicine that Singh and his team are exploring as a promising endotoxemia treatment.

Binding Power of Nanotubes
Singh’s study, funded by the Heather Ryan and L. David Dubé Veterinary Health and Research Fund, involved a series of experiments using cultured mice cells to investigate their interaction with the rosette nanotubes.

The team discovered that the nanotubes bind to the neutrophils, preventing them from crossing a barrier such as a blood vessel. They also found that the nanotubes increased the death rate of neutrophils. The same results were achieved after testing the nanotubes with bovine cells and equine cells, providing evidence that the nanotubes could potentially reduce the horse’s immune response to endotoxins.

“The biggest worry is that you do an experiment in mice that works, but it ends up not working in larger species such as humans or horses,” says Singh. “Our results so far are very encouraging as it seems these nanotubes are working across species.”

The next step had the team performing live mouse studies using multiple routes to administer the nanotubes including inhalation, injection under the skin and intravenous injection. Results are still being analyzed and a publication is in the works for this complicated portion of the study.

Singh hopes to be working with the nanotubes in the live horse by next year, something he’s been looking forward to for a long time.

“One of the major difficulties for studying a treatment on a large scale is getting the funds to do it,” says Singh. “Thanks to the Ryan and Dubé Fund, we’ve been able to think this big. Otherwise we wouldn’t even be able to dream of applying this nanotechnology work to the horse system.”

With researchers around the world exploring new treatments for human sepsis, Singh sees his work being applicable to human medicine as well. He hasn’t tested the nanotubes with human cells yet, but Singh and his team wish to get to this stage eventually.

“This study has been very challenging and exciting,” says Singh. “And the bigger the challenge, the more excited I am. So I feel very happy. It’s a very good team and I hope we can make some advances into understanding and treating endotoxemia.”

For more details, read the Autumn 2011 issue of Horse Health Lines, news publication for the Western College of Veterinary Medicine’s Equine Health Research Fund.

Robyn Thrasher of Edmonton, Alta., is a second-year veterinary student at the WCVM. Robyn produced stories about the veterinary college’s clinical services, research program and its researchers as part of her summer job in research communications.

What’s a rosette nanotube?

A rosette nanotube is an organic, water soluble, biodegradable tube about three to four nanometres (nm) wide. It can be made as long as necessary and it basically looks like white powder in a tube. The tube was designed by Dr. Hicham Fenniri, a professor and senior research officer at the National Institute for Nanotechnology in Edmonton, Alta.

The core of the nanotube consists of bases that form DNA and one can attach various small peptides (proteins) on the outside of the tube, depending on the tube’s purpose.

In Dr. Baljit Singh’s study, the team attached multiple peptide side-arms to each tube consisting of three amino acids – arginine, glycine and aspartic acid.

The nanotubes were dissolved in water and mixed with the cells in a growth medium. The mixture was placed in a chamber with a chemoattractant – a substance that attracts neutrophils. Filter paper between the mixture and the chemoattractant acted as a barrier – similar to the wall of a blood vessel.

What Singh’s research team discovered is that the nanotubes’ specialized peptide side-arms bind to integrins – proteins present on the surface of neutrophils – and prevent neutrophils from crossing the filter paper. With that knowledge, the researchers began looking at how the cells interact with the nanotubes in the presence of endotoxin.

“Normally, neutrophils become activated by endotoxin and will migrate into affected tissues to find the source of the infection,” explains Singh. “But the nanotubes cover the integrins on the outside of the neutrophil, preventing it from leaving the blood vessel and entering the surrounding tissue.”

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