In the age-old battle between man and microbe, people have tried in countless ways to keep their surroundings germ-free, ranging from plain old scrubbing, heat sterilization and chemical disinfectants to high-tech solutions like irradiation or drug-eluting coatings.
Now a new approach could make it easier to keep disease-causing bacteria from forming noxious invisible biofilms on surfaces.
Coatings made from these structures, technically known as "conjugated polyelectrolyte capsules," have proved their mettle in the laboratory against a variety of bacteria and even some spores, raising the possibility that they could be effective against virulent strains, like anthrax, says biochemist David Whitten, associate director of UNM's Center for Biomedical Engineering.
"We also think these should be equally effective against ordinary bacteria and the so-called antibiotic-resistant bacteria," Whitten says.
Unlike some other attempts to use nanotechnology to fight microbes, the microspheres behave like catalysts, meaning they aren't depleted in the bacteria-killing process. "These are essentially permanent coatings," Whitten says.
Remarkably, the spheres only seem to exert their properties against bacteria and spores. "At least in our initial studies, we found that these polymers that are active against bacteria are not killing mammalian cells, which is very nice," he says.
Whitten has also found that the spheres make an effective germicidal barrier on cotton and nylon-cotton fibers, suggesting that they could one day be used to make antimicrobial clothing, disposable bandages, baby swabs and curtains.
"I've been thinking about health applications," says Whitten, who thinks the spheres could even be incorporated into paint and applied to the walls of hospital rooms and commercial kitchens. In sterile settings, where people might track in bacteria on their shoes, "I could see where you might have disposable shoe booties that people could put on," he says.
Other teams have experimented with germicidal microspheres of varying sizes in recent years.
At Brown University, scientists created iron oxide particles just 8 nanometers in diameter that can be guided to the site of an infection in the body with magnets and activated. The metallic particles penetrate bacterial membranes, killing them.
Meanwhile, researchers at Auburn University reported that they had used minute super-strong structures, called single-walled carbon nanotubes, to attach a natural cell-busting enzyme called lysozyme to surfaces, creating a durable germicidal barrier.
Still other approaches include biodegradable microspheres that release antibiotics over an extended period or using spheres to deliver silver nanoparticles that can disrupt bacterial cell membranes.
Whitten has been working with his water-soluble conjugated polyelectrolytes for some time. "These are referred to by many people as [electrically] conducting polymers," he says. "They're really fascinating compounds." While on staff at Los Alamos National Laboratory, he and a colleague started a business to develop bio-sensing units based on these polymers.
They began to realize that these molecules might have antimicrobial properties because of the positive electrical charge that they carry. "We know that bacteria for the most part have a negative charge," Whitten noted.
After relocating to UNM in 2005, Whitten received funding from the Defense Advanced Research Projects Agency, enabling him and his colleagues to whip up a batch of the polymer. "We found if you take a solution of these polymers and add that to a suspension of bacteria, the bacteria rapidly become coated with the polymer," he said.
A year ago, having established that the polymers worked in solution, Whitten and his colleagues decided to see if they could layer them over a template and build three-dimensional microspheres out of the material.
The technique, developed by various research labs over the past half dozen years, starts with a tiny template of manganese carbonate 5 microns in diameter (about one-twentieth the width of a human hair). The polymer is deposited on the template in four oppositely charged layers.
Then the spheres are dipped in a solution that dissolves away the manganese carbonate. Viewed under a microscope, they look a little like deflated whiffle balls. The manufacturing process leaves tiny holes in the spheres through which the bacteria can enter, but not exit, Whitten says.
The polymers were first tested on E. coli bacteria and on the Sterne strain of anthrax spores (which are thought not to be harmful to people). "Those were both killed by the polymers," Whitten said. Time-lapse fluorescent microscopy shows free-swimming bacteria concentrating around the microspheres, where they die.
In a paper published in the journal Applied Materials and Interfaces last year, Whitten's team reported that the conjugated polyelectrolytes were effective against Pseudomonas aeruginosa, a so-called Gram-negative bacterium that is the second leading cause of infection in hospital intensive care units.
The original tests showed that these polymers have significant bacteria-killing properties in darkened laboratory vessels. But they kick into overdrive when exposed to light. "As soon as you hit them with visible light, the bacteria are gone," Whitten said.
"It's pretty clear there's a dark process where this coating interacts with the bacterial membrane and eventually breaks it down," he said. "The polymer is then very close to the inner part of the bacteria. This process in the dark can eventually kill bacteria."
Exposure to light is thought to excite the polymer into releasing singlet oxygen, a highly reactive molecule that more rapidly kills the bacteria, improving the germicidal efficacy to 99.99 percent in some cases, Whitten said.
Meanwhile, each microsphere seems to act a little like fly paper for bacteria, with seven or eight bacteria adhering to it. "We're trying to understand why these capsules are so effective," he said. "[They] seem to ... exude almost like tentacles and they tend to trap the bacteria."
The process tends to leave behind a plaque of dead bacteria. Now the team is looking at thermally activated chemical processes to clean the coated surfaces so that they can continue doing their work, he said. They are also experimenting with larger microspheres made with coated silica, which could collect more bacteria and perhaps be useful for things like water purification, he said. One of Whitten's collaborators is also trying to modify the polymers so that they release even more reactive oxygen molecules.
Whitten is optimistic that because the microspheres kill bacteria in a unique way, germs may be unable to evolve immunity to them. "Most bacteria that develop antibiotic resistance either find a way of deactivating the antibiotic, or more often, just getting it out of their system really quickly," he said. "I don't think either of those strategies would work with this."
Whitten and his collaborators have filed for several patents since launching their research. Meanwhile, some biotech companies have expressed interest in licensing the technology. Whitten says he's content to stay in academia.
"I've started one company," he said. "It was exciting. It was really fun. It was exciting, but I'm too old to do this again. But I would love to be involved in an effort to take this forward."
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