DETOXIFY lNG NERVE AGENTS Academic, Army scientists join forces on enzymes anchored in foams and fibers

bls Ember CGEN Washington

I maghe an enzyme anchored in p l y - mer sc&oldmg that retains its catalyt- ic activity. Imagine this enzyme-

polymer array fnshioned into sponges that can be used to destroy nerve agents on a variety of surfaces, including human skin and tIlc outer Iayers of protective clothing, or into filters in gas masks that can detoxify chemicd agents before a soldier bre:iches them. The Anny has, and so have some academic researchers.

Indeed, the Anny envisions enzymes playing a major role in decontamination systems that would be less toxic, less corrosive, and more environmentalIy benign than those currently in its arma- mentarium. A most promising scheme the Army is stlidying is one developed at the University of Pittsburgh that in- corporates into polyurethane foam nerve agent detoxifying enzymes sup- plied by Texas A M University, CoIlege Station.

Keidr E. LeJeune-a grad~~at t student at Carnegie MelIon University, Pitts- burgh, working in chemical engineering tlrofessor Alan T. Russell's laboratorv at the University of Pittsburgh-has been able to immobilize phosphotriestense in a polyurethane foam matrix and thereby significantly enhance the en- zyme" productivity. That is, the f w d enzyme, stable at mom tempemnlre, "re- tains a significantly high level of activity over s long period of time," LeJeune cxpInins.

After linking phosphotriesternsc co- valently to the foal11 nlatrix, LeJeune 5 able to show that the siill.active enzyme a n eficiently lyctrolyze tlie organophos- phate bond in the pesticide panoxon, a nerve agent analog. Russell's lab is not certified to test the enzyme-fom system against live nerve agents, but pl~osphotri- rstcffisc is alrcndy known to detoxify sa rin and other so-called Gseries nerve agents. althounh not as efficiently its it

ers calculate that 2.5 kg of immobilized phosphotriesterase is probzbly enough to degrade 30,000 tons of nerve agent in one year.

LeJeune has been perfecting the phos- photriesteme-foam system in ElusseIl's iab during the past tl.lree years. For a Ionger period, RusselI has been seeking ways to increase enzyme stability in or- ganic solvents. In fact, kJeune's studies are a namnl outgrowth of work Russell and colleagues have been doing to make protein plastics with the enzyme subtili- sin. The mbtilisin studies and teJeunets work have been funded by the Army Re- search Ofice (ARO), which underwrites basic research.

Robert J. Campbell, associate director for biological sciences in ARO's physicd directorate, describes the enzyme-foam work as "redly unique basic science that is highly relevant to the rnilituy." In- deed, Army scientists at Walter Reed Amvy Institute of Research in Washing- ton, D.C.; the Natick Research, Develop ment & Engineering Center in Massacl~u- setts; and the Edgewood Research, De- velopment, W Engineering Center and the U.S. Army Medical Research Institute

of Chemical Defense (USAMRZCD) in Maryland have been sufficiently intrigued by the promise of the system to collabe rate with Russell's lab on potential mili- tary applications,

One avenue being explored could Iead to a sponge or wipe to cleanse prw tective clothing and skin exposed to nerve agents. The thinking goes like this: A soldier fighting in a chemical environ- ment is wounded by a conventionaI weapon, Before the wound can be treat- ed, medical personnel first have to de- contaminate the soldier's protective gear and his wound arm.

Decontamination today means using water and bleach. But bIeach is toxic, caustic, and not especially environmen- tally friendly. LeJeune and Russell's em zymefoam system could overcome the disadvantages of bleach if, in sponge form, it could be shown to be effective on skin and clotlLing.

LeJeune has tailored poIyurethane foam sponges containing the enzymes acetylcholinesterase (ACW and butmfl- chohesterase (BuChE) to the specifica- tions of Bl~t~pendn P. Doctor, director of the division of biochemistry at Walter Reed. These enzymes govern nesve-tm nerve tmnsmissions in humans and are key targets of org;mophosphorus nerve agents.

In a series of in vim experiments dur- ing a twoadme-half-year period, Doc- tor has shown that the enzymes are bound to the polymer matrix at high effi- ciencies and that they remain catalyticd- ly active after binding.

Doctor tells C&EN that in sponges I "stored in dry form at 4 "C, the e w e s

- . does pamoxo~. The Pittsburgh research- I LeJeune (left) and Russell are developing an enzymebam system wlth milltaw applicafiions.

remain stable for at Eeast two years- there is no loss of activity." By accident, someone in his lab stored a sponge overnight at 75 "C and the enzymes lost only half of their activity, he says. This me:ms that the sponges co~rld possibly be used in a desert environment, n prime considenition since the 1991 Per- sian Gulf War.

Using the nerve agent simulants pam- oxon itnd diisapropyl fluoropIiosphate, Doctor 1x1s also shown tlmt AChE and BuCllE "are inhibited by the surrogates in a quantitative manner and fire reacti- v;ited by an oxime." Unlike p11osphotri- esterase-which hydrolyzes G-series nerve agents and their toxic surrogates to more benign by-products-the en- zymes are themselves inhibited by nerve agents and their surrogates. But in the short period before AChE and

Phosphofrlerterase ( leff] and acetylcholinesterase (below left) have been Immubilized En polyurethane foam (below] by the polymerization reaction (bottom) to produce fabrics suitable for prof eetive garments.

With Doctor's proofofconcept experi. m a t s nearly completed, the next step is to test the enzyme-impregnated sponges against actual nerve agents, which Doc- tor's lab is not geared to do. Instead, Dm- tor wiIL coIlaborate with researchers in David E. Lenz's labs at U W C D m ex- pose the sponges to the M e s nerve agents sarin and soman. Lenz, chief of

I I t i e biochemical pharmacology branch at USAMRICD, says he's "pretty sure the

1 I sponge is going to work with sarin, but soman is more problematic." With so- man, there is less time to reactivate the

OCN,

enzyme. Within the next two

years, Doctor's and Lenz's Enzyme PNCO Iabs will begin testing the

enzymefoam decontasnina- tion system on animds. If

I the sponges are found to be safe and effective in ani-

0 NCO mal models, Lenz explains ~nzyrne-~-g-#bl~~~ that the Amy would "then go to the Food Sc Drug Ad- ministration for approval for use."

BuChE are permanently inhibited-that is, dealkylated-they can be reactivated by a nucleopl~ilic oxime. To take advan- tage of this window of opportunity, U.S. troops in the Gulf War carried two prophyIactic drugs against nerve agent exposure: atropine and pralidoxime chIoride, also called 2-PAM chIoride. These substances have to be used in tan- dem and very quickly after exposure to nerve agents.

Doctor is still carrying out enzyme re- activation tests, but data to date indicate that dipping a sponge that has been ex- posed to a nerve agent surrogate 'in a buffered oxime solution will reactivate the enzyme and revive the sponge," he says. There is only a very small mechmi- cal loss of enzyme after repeated reacti- vations, he adds. The ability to reuse the sponges makes them cost-effective and adds to their advantages over water and bleach.

"From a medical point of view, these sponges hold a very good promise of be- ing a usefill tool in deconraminating pea ple in a civilian or miIitay environment," Doctor contends. Wlde military person- nel-suited up in protective gmr-have n modicum of protection against nerve agents, civilians, lacking sudi protection, do not. Since the 1975 win attack in the Tokyo subwy system by a Japmese cult, the development of a decon~unination system lor civilian use has taken on in- creasing importance.

The initial focus of these medicaI studies has been on detoxifying the area around a wound and the protec- tive clothing worn by an injured soI- dier. But Lenz specuIales t l ~ t the spong- es could be considered for "whole b d y deconmmination," that is, treating all ex- posed skin on an unprotected soldier or civilian.

In addition to the sponge form, the Army researchers suggest that if the poIy- urethane-immobilized enzyme is pre- pared in granular form and incorporated into topical skin creams, such c m s might be able to detoxify nerve agents. It's nor so piein-the+ as it might first sound. The Army is now trying to get FDA approvd for an inert barrier cream that contains no enzyme,

For several years, km's lab has been working on developing an enzyme from humans that could, acting as a biological scavenger, protect military personnel against the toxic effects of chemical agents. To that end, the lab has done site- directed mutations on BuChE and has found a mutant that 'no longer is inhibit- etl by orgnnophosphoms nerve agents but which actually c;ltnlyl,es the hydroly- sis of the agents," Lenz explains. This mutated enqme no longer requires an oxime for reactivztion.

So, why not uke this mu~~teted enzyme and insert it into the poly~~rethme f o m matrix? If successft~l, a sponge could be fashioned that wouIdnt teed to be

s m n m is, 1997 cw 27

dipped into an oxime solution to remain fi~nctionaI. Lenz says this concept is be- ing considered, although implementing it is years away. "We have the h m n material, the enzyme in a stable exprep sion system. Now it's a matter of makiig enough of it to supply to someone like R~~ssell who can immobiize it onto a sol- id support."

But before that can happen, the rnu- meed BuChE has to be tweaked a bit. AF- though it is capable of catdyzing the hy- drolysis of nerve agents, Lenz says, "the rate of hydrolysis is not sufficient to make it a viable candidate for protection. Hence, we continue our studies to irn- prove the catalytic properties of the mu- tated enzyme."

Lenz cautions that the operational needs of the various branches of the armed forces wiII ultimately determine how and in what form stn enzyme-foam system is used to treat wounds and skin. Of course, use on humans is dependent on getting FDA approval.

In addition to its medical applications, the enzymefoam matrix can be made into fabrics that have the potential for use h protective garments.

The c w e n t threelayer protective suit weighs about 6 Ib and consists of an out- er layer of cottonfnylon twill, a middle layer of activated carbon in poIyurethane foam, and an inner, Iigbt fabric that lays against the body. The mechanism of pm tection is simpIe and efficient: absorp- tion of toxic compounds by the activated carbon.

Activated carbon, however, is rela- tiveIy heavy, so the Army is seeking ways to reduce the weight and thirk- ness of the garment. In ttleory, the acti- vated carbon layer could be replaced with catalytic enzymes encapsuIated in a thin (short path length) polymer sys- tem that would allow for high perme- ation rates of the reactants.

Late Iast year, LeJeune sent his en- zyme-impregnated foam to Dondd Rv- in's Iabs at the Army Natick Research, Development & Engineering Center for testhg. This lab has long been sarclling for "materials that act as barriers to toxic vapors," explains Rivin, principal scien- tist In s~trvivability.

fivin says his labs have been able to confirm that phosphotriestense "is ac- tive against the simulant nerve agent panoxon" when the pesticide is deliv- ered in liquid form. That is, there is a high rate of enzymecatalyzed conversion of psnoxon to pnimphenol.

To allow Rims Iabs to test the en.

Doctor: sponges as useful tools

zyrne-foam system against toxic vapors, In jeune incorporated phosphouiesterase into the poIylmthane structure; polymer- ization took place on a fabric st~ppIied by Rivin. k jeune also supplied his more "normal" thin Payer of enzyme-impregnat- ed f o m over which a cotton/nylon rdl was placed before testing, 11e mil[ consti- tutes the outer Ixyer of the current tlu-ee- layer protective suit.

To form the vapor, Iiquid droplets of pamoxon are pIaced on the twiZE fabric on top of the customized enzymefom, and air, humidified air, or nitrogen is passed tlmugh the droplets. If the en- zyme is active it will convert the panox- on vapor to pnitrophenol. And, indeed, enortgh of the by-product has been re- covered in the foam Iayer "to keep us in- terested in continuing to try to optimize" the system, Ilivin teUs C&EN. The n t e of conversion, however, is not as high as it was when the enzyme in foam was chal- lenged with liquid paraoxon.

Test resuIw have been more incon- clusive when vapors of diisopropyl fluo- rophosphate @FP), another surrogate for the G-series nerve agents, chal- lenged phosphotriesterase in the vari- ous fabric constructions. "We didn't see a high rate of conversion, and we are not stwe if we did the experiments cor- rectly," Rlvin says. Phospl~otciestense is not as active against DFP as it is against pmoxon, and "it is difficrtlt to get a con- trolled, low volatility vapor concentra- tion," he explains. The experiments are being repeated.

Rivin says tlre Russell-kJeune system "has goad stability, high reactivity, and there is little loss of enzyme activity,"

and it is a good candidate for making the protective suit lighter in weight and thickness. But it is not the only way to do so.

Another avenue is to put powdered cros l i i ed enzyme crystaIs capable of hydrolyzing nerve agents benveen two Iayers of fabric or foam. Rivin is cofIab@ rating with Alttls Biologics, Gunbridge, Mass., in demonstrating the feasibiiity of this system.

Still another approach uses nanofibers produced by electrospinning. Electro- spinning employs electrical forces to prc, duce b n g poIymer fibers with ~ d i a e t m ranging from 40 to 2,000 nm. The weav- er of these very small fibers in Rivin's labs is Heidi L. Gibson, a poIymer re- searcher. "Electrospinning produces R bers that are IO to 100 times h e r than common textile fibers," Gibson explains. "They look like cobwebs."

She 5 trying to incorporate catalytic enzymes into these cobwebs "to pro- duce microporous, membranelike mate- rials with a very high surface area." f i e high surface area is needed for good re- activity and transport properties-and for the wearer's cornfort, she says.

Gibson is working with an organ* phosphonrs hydrolxse-not phosphotri- esteme-as well as an inorganic catalyst and copper chirosan. And she is still searching for t l ~ e appropriate fiber into which to incorporate the catalyst. The fi- ber must be robust in the wpor chal- lenge test and be able to absorb mois- ture. "But so far," Gibson bemoans, "the fibers have been dissolving."

Nanofibers offer the advantage of a wider palette of polymers than can be made into foam. And, because the fi- bers can actualIy be woven into che clothing fabric, the activated carbon Iay- er might be eliminated entireIy, thus de- creasing the weight of the protective suit ~ i g ~ a n t l y .

No matter which approach to modify the protective suit is wentuxlly chosen, its key feature will be that it produces very thin fabric layers capable of incor- porating a mixture of enzymes. An en- zyme mix is needed to protect the sol- dier against mustard gas and other chem- ical warfare agents, not just nerve agents, Kvin expIains.

The gas mask is another component of protection against chemical warfare agents. And RusseU tells C&EN that hi lab "is looking at enzyme activity with at- mospherically delivered substrates." He gaes on to say that his lab has been able to show that phosphotriesterase "works

28 SEPTEMBER 15,1997 C&EN

on a gasphase substrate, so theoretically show that some of their dipeptidases it could be used as a filter in a gas mask or as a filter in a stack of an incinention plant."

The enzymefoam matrix may ulti- rnalely be found not to be a suitable re-

thane, grind up the enzyme-ply- a prefdter in front of the charcoal to de- a fme powder, and incorporate

lenging to stabilize enzymes in the air- water interface" found in fire-fighting

my's Edgewood Research, LeJeune also thinks it might be possible

prefilter may provide Ionger life for the to replace or reduce the bleach in German gas mask filter." emulsion-which is used to decontarni-

DeFrank's group 5 trying to deveIop enzyme-based solutions for decont;uninat- ing large surface areas-Melds, ports, battlefields, and dviIian facilities. The Russell-LeJetme solid, immo- L i e d enzyme foams are not suit- able for decontaminating these large, Futed ares , where enzymes , + ' delivered in liquid sprays could work. But DeFank believes that .

could be used to wipe small sw- face arms clean of chemical war- fi~re agents. He is especially chink- be lessened and the area decom ing of confined spaces like those tarninated might be enlarged, he in aircraft cockpits where a h o ~ ing down with a water-based de- toxifying solution is not appre two decontamination schemes, priate. And 11e is considering ek LeJeune is also planning to in- - &me-containing polyuretiane Gibson (fop) uses electrospinnlng to generate cobweblike corporate othe; enzymes into

for the decont;uninntion nanofhers that can Incorporate cata?ytlc enzymes. the polymer skeleton. As he ex- of equipment, especidy sensitive electronics. stn~ction metl~ods are being considered.

Eventually, D e F m k says, "I would In fact, DeFnnk's group has recently like to see if our enzymes work in Rus submitted a proposal to the Army's sell's system." DePnnk's group works Chemical & Biological Defense Corn- with dipeptid~tses active against G-series mand, seeking funding for studies on nerve agents, not pl~ospl~otriesterase. "enzyme-catalyzed destruction of the "Ideally, our decontaminating systems agents as an alternative to incineration," would have to have a mixture of en- ERDEC spokesman James M. Allinghm zymes-dipeptidases, phosphotriester- says. No funds have yet been allocxted ase, DFPme-:md other substances to be for this approach. effective against nerve agents, mllstard From the destruction of weapons to gas, and biological agents. " the fighting of fires mq seem a stretch,

AIthough decontnmination i s the but enzymes could play a role in bath ac- thnzst of DeFnnk's effarts, it is conceiv- tivities. Army tmks are limited in cargo able that inlmobillzed enzymes co11ld be space, so it wo~dd be advantageous if used to destroy sanle of the chemlcP fire-fighting vehicles could simuItaneous stocks tbe U.S. now stores at various Iy spray a singIe compotlnd that could sites. In rniIitnty parlance, the destmc- fight fires and decontami~iate surfaces tion program is known as demilitariza- exposed to nerve agents. This might be tion. Incineration of weapons and their possible if catalytic enzymes could br: in- chemical agents and neutmlization of corpomted into fire-fighting foams, md ~ W S L V ~ g;& a ~ l d VX nerve gas stored in DcFnnk's group and Lr:Jeunt in R~~sse[I's bulk are the current destruction teclmol- lab are testing tllis concept. ogies favored by the Army, but other d e DeFrank's group has been able to

plains, phosphotriesterase i s extremely active against pmoxon, but 'it is not very active against more toxic agents" such as sarin. A squid enzyme, DFPase, is effective against sarin and other G-series nerve agents, so he in- tends to incorporate it into polyure- thane to see if he can get enhanced p m ductivity-as he did with pl~osphotries- teme.

With these variations on a theme, it would seem as if the enzyme-foam work has been pIayed out. But Russell demurs, saying, "No. Probably not. We don't need to do a lot more to make it more practicable, however." Still, his 1x6 contint~es to Cm-tune phosphotries- tense, attempting to enhance its stabil- ity in systems like German emulsion, for instance.

It may turn out that the versatile scheme devised by Russell and kJeune is limited only by what creative minds can imagine, cIever hands manipulate, md p l e n t u h d i n g sappo*4