Immaculate standards for surgical cleanliness allow antibiotics and the patients’ immune systems to handle bacteria that get into most surgical sites, but with multi-antibiotic-resistant strains on the rise, doctors are interested in methods that could completely sterilize wounds.
While chemical antiseptics help kill bacteria, concentrations that totally eradicate them could seriously aggravate the already-damaged tissue. But in the last few years, a new antiseptic has emerged that is much gentler toward mammalian tissue, annihilating the bacteria while accelerating the healing of the wound. It’s not a liquid, gel or paste – it’s a plasma.
Plasma is a glowing gas containing free electrons, charged atoms and atoms with excited electrons. In nature, plasmas typically exist in extremely hot environments, such as a bolt of lightning or the sun. The group of John Foster, an associate professor of Nuclear Engineering and Radiological Sciences, specializes in making plasma by giving smaller amounts of energy directly to the electrons, avoiding temperatures that could vaporize human flesh.
“Those free radicals always attack something, break something up,” said Raoul Kopelman, the Richard Smalley Distinguished University Professor of Chemistry, Physics, Biophysics, Biomedical Engineering and Applied Physics, and Foster’s collaborator. “There is a good chance it will get rid of bacteria.”
Plasma-produced radicals are thought to be beneficial for healing because, in addition to breaking up the cell membranes of bacteria, the nitrogen oxide echoes the body’s own call for repairs.
“It’s like calling the fire department, police department and a nearby town’s fire department,” said Foster. “This precursor radical essentially initiates the healing response of the body.”
Studies have shown that plasma encourages mammalian cells to grow and spread, and nitrogen oxide plays an important role in guiding the formation of new blood vessels to feed the regenerating tissue. Still, the details of what plasmas do to cells are largely unexplored. To discover these effects, Foster’s team is bringing their plasma beam to Kopelman’s lab.
The collaboration is starting by applying plasma to cancerous human cells since they tend to be hardier than healthy cells. Foster’s team makes the plasma by flowing helium through a quartz tube with one electrode running through the center. The other electrode coils around the end of the tube, creating a powerful electric field when the electrodes are charged up to about 2,000 volts.
The brief, high-voltage pulses from the electrodes accelerate the few loose electrons in the gas so that they ram helium atoms with enough energy to free more electrons. Those electrons then strike other atoms, and so on. This process also boosts electrons in the atoms into energetic states, causing the plasma to glow as these atoms release the energy as light.
When the avalanche of electrons reaches the air, it creates those reactive molecules in a blue-white shaft of captive lightning. Unlike the plasma of natural lightning, it is at room temperature – only the free electrons are hot, and they’re too small in mass and in number to make much difference. “You could put your hand in it,” said Foster.
But you wouldn’t want to leave your hand in there for long. At higher doses, the plasma coaxes cells into undergoing programmed cell death, the body’s way of getting rid of unwanted cells without inflaming the surrounding tissue. Other researchers have used plasma to remove tumors by killing off the cells. Foster’s and Kopelman’s work may clarify where the line between tissue healing and cell death lies for human cells exposed to plasma.
Their first experiments, in April, measured changes in the cells’ internal acidities. Preliminary results suggest that plasma pushes the cells toward higher acidities, which may affect their growth. Kopelman and Foster intend to measure the pH levels within cell compartments and study the concentrations of reactive molecules containing oxygen. With this information, they can begin to understand the mechanisms that allow low doses of plasma to kill bacteria while healing mammalian tissue.
Although plasma seems to be good for sterilizing wounds, it’s not suitable for maintaining bacteria-free surfaces. That might be a job for frog-inspired coatings.
If you synthesize peptides chemically, just one of them may cost a couple thousand dollars… So we use nature to do the legwork.Erdogan Gulari
Tubes, catheters and other artificial points of access into patients’ bodies allow the direct delivery of drugs and nutrients, help with breathing and more, but these conduits also pose a risk. If bacteria can get into them, they have a shortcut past the patient’s defenses. Erdogan Gulari, the Donald L. Katz Collegiate Professor of Chemical Engineering, believes that these devices could be made to fight off the bacteria with a coating of antimicrobial molecules.
When humans get sick, we usually absorb viruses or bacteria through mucus membranes, such as the moist tissues of our eyes, noses and mouths. These sites are reasonably protected, but a frog’s skin is like one big mucus membrane. So why aren’t they sick all the time?
It turns out that frogs produce a huge range of microbe-killing peptides, which are small protein-like molecules, on their skins. These peptides open holes in the cell membrane of a bacterium, killing them. Humans use antimicrobial peptides as well – some immune cells that swallow bacterial invaders slay them with peptides.
Gulari’s team is developing a way to tweak natural peptide designs to fight target microbes more effectively. Because designing peptides is still a trial-and-error affair, this means producing and testing thousands to millions of them.
“If you synthesize peptides chemically, just one of them may cost a couple thousand dollars, whereas if you use a yeast or E. coli cell to synthesize them, you can get tens of thousands of them at almost no cost,” said Gulari. “So we use nature to do the legwork.”
They started with the peptide Plantaricin-423, which is harmless to humans but capable of killing the food-borne pathogen Listeria. Peptides and proteins are strings of molecules called amino acids, and Plantaricin-423 contains 37 of them. The first 18 bind the peptide to the target cell membrane. Since Plantaricin-423 already attaches to Listeria but not human cells, Gulari’s team left those alone.
To make the peptide deadlier, they changed the 19-amino-acid hole-poking section. A computer program randomly assigned one of six amino acids to each spot, with the option to shorten the chain by one or two amino acids. That resulted in about 12,000 different peptides. They then made DNA blueprints for each of these designs and slipped them into E. coli cells, where the bacteria’s machinery could begin churning out peptides.
“We can do large petri dishes of 4,000 or 5,000 colonies, so that’s 4,000 or 5,000 different peptides being produced,” said Saadet Albayrak Guralp, a research associate in Gulari’s lab. With those production levels, she isn’t fazed by the idea of testing hundreds of thousands of peptides, up to around a million.
To find out how well the peptides worked, the team grew the Listeria on the same petri dish. The laboratory E. coli bacteria were designed with leaky cell membranes, so the peptides escaped into the petri dish, creating Listeria-free zones around the E. coli colonies.
The sizes of those zones gave Gulari’s team a general idea of how well the peptide discouraged the growth of the Listeria, but that measure is dependent on how well the E. coli hosts produce the peptides. The group isolated the most promising peptides to find out how potent they were at killing the Listeria. It turned out that the best could keep the bacteria at bay with half the concentration of the original Plantaricin-423.
This first study was stage one of finding the best Plantaricin-inspired anti-Listeria peptide, Albayrak explained. “As soon as we start seeing a pattern of increased activity based on mutations, then we can really focus on that part [of the peptide],” she said. “It’s like screening and designing.”
She and Gulari hope that the method may also uncover peptides that can fight off other hospital bugs such as staph and those that cause pneumonia in patients on ventilators.
But no preventative method is completely successful, so doctors will continue to deploy antibiotics to fight infections. Designer peptides may become new drugs as well if they can be coated so that the body doesn’t break them down before they reach the infection. But new antibiotics aren’t the only options – another team is exploring ways to make the current antibiotics work better.