By Liz White, Editor
Scientists at the McGowan Institute for Regenerative Medicine at the University of Pittsburgh have recently synthesised a polyurethane/polyacrylate material that could offer a breakthrough in protection from terrorist attacks. The polymers contain functional groups that can deactivate both biological and chemical toxins, as described recently in a paper in the journal Biomaterials.1
Weapons of mass destruction could contain biological agents such as the smallpox virus, or chemicals such as the nerve agent sarin, said senior investigator Alan Russell, professor of surgery, University of Pittsburgh School of Medicine, and director of the McGowan Institute. “That uncertainty calls for a single broad-spectrum decontamination material that can rapidly neutralise both kinds of threats and is easily delivered or administered,” he said.
“Typically, labs engineer products that are designed to serve only one narrow function,” Russell added. “Our lab applies biological principles to create materials that can do many things, just like our skin protects us from both rain and sun,” he added, in a press statement from the Institute.
The team has devised a novel polyurethane fibre mesh containing enzymes that lead to the production of bromine or iodine, which kill bacteria, as well as containing oximes that generate compounds that can detoxify organophosphate nerve agents.
“This mesh could be developed into sponges, coatings or liquid sprays, and it could be used internally or as a wound dressing that is capable of killing bacteria, viruses and spores,” commented lead investigator Dr Gabi Amitai of the McGowan Institute and the Israel Institute for Biological Research. “The antibacterial and antitoxin activities do not interfere with each other, and actually can work synergistically,” he said, in the statement.
In their experiments, the material fended off Staph aureus and E. coli, which represent different classes of bacteria. And after 24 hours, it restored 70 percent of the activity of acetylcholinesterase, an enzyme that is inhibited by nerve agents, leading to fatal dysfunction of an essential neurotransmitter.
Discussing the work in a 29 March telephone interview, researcher Richard Koepsel said the team at McGowan has “tried to make defensive surfaces for quite some time,” mainly working on polyurethanes and polyacrylates, to give materials which are able to kill bacteria statically.
“We’ve looked at immobilising enzymes which specifically react with chemical agents, in the past,” and the team has over 20 years experience of working with PUs, he added.
This project combined the two types of work: “Our understanding of enzyme immobilisation — and to a certain extent we also relied on Gabi Amitai’s understanding of how oximes are involved in catalytically degrading the chemical agents.” With PU, the Institute has done, “multi-point immobilisation, so the enzyme doesn’t get released at all. It is still able to do its catalytic work in the context of the PU matrix.” In the current case, though, the enzymes are not deliberately covalently bound.
“The way we made these matrices this time was with electrospinning (ES),” Koepsel said.
Spinning a fine fibre
Essentially, ES involves using a metal tube with a small outlet, which is charged, as is a target, about 20 cm away. “We pump the PU in solution through the metal tube which charges all the molecules. As they come out of the end of the tube, the voltage drop attracts them to the target,” Koepsel said. As it exits the tube, the material “whips around in space” to make fibres, depositing a fibrous mat on the target (the charge gap is between 20 and 30 kilovolts).
During the spinning process, the enzymes, in solution, are added in a second stream, as is the polyacrylate polymer that has the oximes on it. “So we can mix the PA with the PU and get a co-fibre, if you will,” Koepsel noted.
Iodine will also work against some chemical agents as well as microbiological ones, he said.
Electrospinning is one aspect that makes this a novel approach, Koepsel said. “When we take it off the target it has the feel of a latex glove.” The other novel part is the combination of activities – to make a broad-spectrum agent against both chemical and biological attack.
Koepsel said the team used Chronoflex AR polyurethane from AdvanSource Biomaterials Corp., of Wilmington, Massachusetts. This medical-grade PU, based on an aromatic diisocyanate, has good tensile strength and flex life. Products are supplied dissolved in dimethylacetamide, and Koepsel said “we cast it into a film and re-dissolve it in another solvent for ES.” For electrospinning, the solvent has to be extremely volatile. This is because to get the fibre formation, the solvent has to evaporate as the material comes out of the ‘spinnerette’ and whips around in space before it hits the target.
At McGowan, the PU was dissolved in hexafluoroisopropanol (HFIP), but other solvents are possible when it is scaled up.
Discussing the next step, Koepsel said: “We think that the technology itself is pretty robust.” Ideally, he said, someone would come along with funding to help develop the process.
The group is currently in discussions over patenting the latest technology, where there are some issues involved, he noted.
The treated PU/polyacrylate has the potential for being formulated into a sponge, a liquid spray or a coating — a number of different types of materials — after it has been spun, said Koepsel. And it has potential for use in wound treatment, he agreed. With a previous material, without the oxime, “we designed that specifically as a bandage, which evolves iodine, as a sterilant,” he explained.
And Koepsel said there is further work to optimise some areas, “to see if there is a better method of incorporating the enzyme. We do lose enzyme activity,” he said, pointing out that, “the HFIP kills the enzyme within minutes, so we have to mix it and spray it and get it out of the solvent rapidly, so that it’s still active.” People are concerned over the threat of an attack with both nerve gas and viral agents, Koepsel said. It’s possible that, “it’s not in the public eye right now — but I think the military is very interested,” he said.
Reference 1. Decontamination of chemical and biological warfare agents with a single multi-functional material, Gabi Amitai, Hironubu Murata, Jill D Andersen, Richard R Koepsel and Alan K Russell, Biomaterials, Vol 31, 2010, p 44417-4425.
New approach to dual attack
The material developed at the McGowan Institute contains halide-producing reagents which act as a biocide and can also disable some chemicals, while for effective action against nerve gas materials — often complex organophosphates (OPs), which attack cholinesterase (ChE) pathways in the body — oximes are known to be a valuable approach.
In these experiments, the team used the oxime N-alkyl 4-pyridinium aldoxime (4-PAM, on an acrylate polymer – dimethylacryamide methyl methacrylate (DMAA-MA).
The enzymes deal primarily with microbiological attack and work by producing halogens, Koepsel explained. Two enzymes are used: glucose oxidase, which oxidises glucose to produce hydrogen peroxide and CO2. The H2O2 is used by the other enzyme, horseradish peroxidase, which can, in the presence of peroxide, take a halide ion and convert it into a halogen.
“So you can take two iodide ions and make iodine, which is a very oxidising compound,” Koepsel said.
The halogen-producing enzymes were shown to be effective bactericides, killing E-coli in one hour.
The PAM agent’s effect on the organophosphate diisopropylfluorophosphate (DFP) was dosedependent, reaching 85 percent in 30 min.
As the team points out, the ability of halides to kill a wide variety of microorganisms, including antibiotic-resistant bacteria, viruses and fungi, “Has been known for centuries.” And they note that this now includes the ability to destroy methicillin resistant Staphylococcus aureus (MRSA).
Meanwhile PAM materials are effective as ChE reactivators after OP pesticide poisoning and nerve gas exposure. In contact with OPs, the research showed they can slowly deactivate agents such as Soman and VX — the two main agents, Koepsel said.
Previous work showed the effectiveness of the glucose oxidase and horseradish oxidase when electrospun into a PU fibre mesh in producing halides that killed S aureus and E coli.
The electrospinning does not create covalent bonds between the non-reactive PU and the acrylate copolymer, so that the water-soluble acrylates containing the oxime can leach out of the polymer matrix. Part of the project involved developing a means of successfully crosslinking the two polymer chains, using benzophenone to give photo-activated crosslinking of the soluble polyacrylate with PU after electrospinning.
